Microspheres useful for therapeutic vascular embolization

ABSTRACT

Provided herein, for example, are microspheres comprising a gelatin or gelatin substitute and a copolymer of a N-tris-hydroxymethyl methylacrylamide monomer unit, a diethylaminoethylacrylamide monomer unit and a N,N-methylene-bis-acrylamide monomer unit. Also provided are methods of producing microspheres comprising a gelatin or gelatin substitute and a copolymer of a N-tris-hydroxymethyl methylacrylamide monomer unit, a diethylaminoethylacrylamide monomer unit and a N,N-methylene-bis-acrylamide monomer unit. Further provided herein, for example, are compositions comprising the microspheres and methods of using the microspheres and compositions thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of now pending U.S. patentapplication Ser. No. 13/890,038, entitled MICROSPHERES USEFUL FORTHERAPEUTIC VASCULAR EMBOLIZATION, filed on May 8, 2013, which is adivisional of U.S. patent application Ser. No. 13/014,172, entitledMICROSPHERES USEFUL FOR THERAPEUTIC VASCULAR EMBOLIZATION, filed on Jan.26, 2011, which claims priority to U.S. Ser. No. 61/460,742 filed onJan. 27, 2010, and EP Serial No. 10305093.6 filed on Jan. 27, 2010, eachof which is incorporated herein by reference in its entirety.

FIELD

Provided herein, for example, are microspheres comprising a gelatin orgelatin substitute and a copolymer of a N-tris-hydroxymethylmethylacrylamide monomer unit, a diethylaminoethylacrylamide monomerunit and a N,N-methylene-bis-acrylamide monomer unit. Also provided aremethods of producing microspheres comprising a gelatin or gelatinsubstitute and a copolymer of a N-tris-hydroxymethyl methylacrylamidemonomer unit, a diethylaminoethylacrylamide monomer unit and aN,N-methylene-bis-acrylamide monomer unit. Further provided herein, forexample, are compositions comprising the microspheres and methods ofusing the microspheres and compositions thereof.

BACKGROUND

Therapeutic vascular occlusion (i.e., embolization) is used to preventor treat certain pathological conditions in situ. It can be administeredby means of catheters making it possible, under imagery control, toposition particulate occlusion agents (i.e., emboli) in the circulatorysystem. It has a variety of medical applications such as in thetreatment of tumors, including, e.g., uterine fibroids, vascularmalformations and hemorrhagic processes. For example, vascular occlusioncan suppress pain, limit blood loss on the surgical intervention tofollow embolization or even bring on a tumoral necrosis and avoid theoperation. In the case of vascular malformations, vascular occlusionenables the blood flow to the normal tissues to be normalized, aids insurgery and limits the risk of hemorrhage. In the hemorrhagic processes,vascular occlusion can produce a reduction of flow, which promotescicatrisation of the arterial opening(s). Moreover, depending on thepathological conditions treated, embolization can be used for temporaryas well as permanent purposes.

Different types of emboli have been used for embolization, for example,liquid agents (e.g., acrylic glues, gels or viscous suspensions) as wellas particulate agents (e.g., miscellaneous polymers, dura mater, gelatinsponges, spheres, balloons or spirals), including EmboSphere® trisacrylgelatin microspheres (BioSphere Medical, Rockland, Mass.) (see also,e.g., U.S. Pat. Nos. 5,635,215 and 5,648,100).

Among the various occlusion agents, microspheres have demonstratedbetter embolic properties over other solid emboli. However, the qualityand yield of the microspheres often varies due to materials used in theproduction process and methods of making them.

Accordingly, there remains a need for methods of producing microsphereswith, e.g., a better yield productivity and possibly better uniformityor purity. In addition, there also remains a need for microspheres with,for example, a better or more consistent quality from production batchto production batch that are capable of providing optimized emboliceffects.

SUMMARY

The methods provided herein relate generally to microspheres andcompositions comprising the microspheres. Also provided herein aremethods of producing and using the microspheres.

Provided herein, in one aspect, is a microsphere comprising: (a) acopolymer comprising a N-tris-hydroxymethyl methylacrylamide monomerunit, a diethylaminoethylacrylamide monomer unit and aN,N-methylene-bis-acrylamide monomer unit, and (b) crosslinked gelatin.In certain embodiments, the microsphere exhibits in a ¹H NMR spectrum, afirst peak from about 3.5 ppm to about 4 ppm, a second peak from about 3ppm to about 3.5 ppm, and a third peak from about 1 ppm to about 1.5ppm. In specific embodiments, (i) the integration ratio of the secondpeak to the first peak is about 0.50 to about 0.65, (ii) the integrationratio of the third peak to the first peak is about 0.55 to about 0.75(e.g., about 0.61 to about 0.75), or (iii) a combination of (i) and(ii). In certain embodiments, the N-tris-hydroxymethyl methylacrylamidemonomer unit is an ultra-pure monomer unit, thediethylaminoethylacrylamide monomer is an ultra-pure monomer unit and/orthe N,N-methylene-bis-acrylamide monomer unit is an ultra-pure monomer.

In another aspect, provided herein is a microsphere comprising: (a) acopolymer prepared by copolymerizing a N-tris-hydroxymethylmethylacrylamide monomer, a diethylaminoethylacrylamide monomer and aN,N-methylene-bis-acrylamide monomer, and (b) crosslinked gelatin. Incertain embodiments, the microsphere exhibits in a ¹H NMR spectrum, afirst peak from about 3.5 ppm to about 4 ppm, a second peak from about 3ppm to about 3.5 ppm, and a third peak from about 1 ppm to about 1.5ppm. In specific embodiments, (i) the integration ratio of the secondpeak to the first peak is about 0.50 to about 0.65, (ii) the integrationratio of the third peak to the first peak is about 0.55 to about 0.75(e.g., about 0.61 to about 0.75), or (iii) a combination of (i) and(ii). In certain embodiments, the N-tris-hydroxymethyl methylacrylamidemonomer is an ultra-pure monomer, the diethylaminoethylacrylamidemonomer is an ultra-pure monomer and/or the N,N-methylene-bis-acrylamidemonomer is an ultra-pure monomer.

In another aspect, provided herein is a method of making microspherescomprising: (a) preparing an aqueous solution comprising (i) aN-tris-hydroxymethyl methylacrylamide monomer, (ii) adiethylaminoethylacrylamide monomer, (iii) aN,N-methylene-bis-acrylamide monomer, and (iv) gelatin, wherein theN-tris-hydroxymethyl methylacrylamide monomer is an ultra-pure monomer,the diethylaminoethylacrylamide monomer is an ultra-pure monomer and/orthe N,N-methylene-bis-acrylamide monomer is an ultra-pure monomer; (b)adding the aqueous solution to a liquid organic phase (e.g., an oil,such as a mineral oil, such as a paraffin oil) that has low miscibilityin water, before or while stirring; thereby producing microspherescomprising a copolymer comprising N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and N,N-methylene-bis-acrylamide monomerunits; (c) crosslinking the gelatin. In some embodiments, the methodfurther comprises subjecting the microspheres to sonication (e.g.,ultrasonication) prior to crosslinking the gelatin. In one embodiment,the aqueous solution is added through a feed ring to the liquid organicphase. Also provided herein are microspheres produced by these methods.

In a another aspect, provided herein are methods of embolization in asubject, comprising administering to the subject the microsphere(s)provided herein.

In yet another aspect, provided herein are methods of managing ortreating an angiogenesis-dependent disease in a subject, comprisingadministering to the subject the microsphere(s) provided herein.

TERMINOLOGY

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications are incorporated herein by reference in their entirety. Inthe event that there is a plurality of definitions for a term herein,those in this section prevail unless stated otherwise.

The term “about” or “approximately” means within 20%, such as within15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%,within 4%, within 3%, within 2%, within 1%, within 0.5% or less of agiven value or range.

As used herein, “administer,” “administration” and “administering”refers to the act of injecting or otherwise physically delivering asubstance as it exists outside the body (e.g., a particle or microsphereprovided herein) into a patient, such as by, but not limited to,pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal,intravenous, intraarterial, intrabiliary, intraocular, intraosseous,intramuscular delivery and/or any other method of physical deliverydescribed herein or known in the art. In specific embodiments, themicrospheres are delivered using a syringe and/or a catheter. When adisease, or a symptom thereof, is being treated or otherwise managed,administration of the substance typically occurs after the onset of thedisease or symptoms thereof. When a disease, or symptom thereof, isbeing prevented, administration of the substance typically occurs beforethe onset of the disease or symptoms thereof. Such administration, incertain embodiments, results in the delivered particles (e.g., amicrosphere provided herein) contacting the target area (e.g., a bloodvessel, tissue or organ).

The term “angiography” refers to a type of x-ray that is done to imageblood vessels in various parts of the body (e.g., liver, prostate,uterine), so as to determine whether the vessels are diseased, narrowed,enlarged or blocked altogether. In certain embodiments, an angiogram(e.g., x-ray) is taken by injecting a contrast material to highlight thevessels, after passing a catheter into an artery leading to the bodyarea of interest. “Superselective angiography” refers to angiographywith the use of a smaller catheter may be passed through a larger oneinto a branch artery supplying a small area of tissue or a tumor.

The term “arteriovenous malformation,” “AVM,” “vascular malformation”refers to a group of diseases wherein at least one (and most typically,many) abnormal communications between arteries and veins occur,resulting in a local tumor-like mass composed predominantly of bloodvessels. Such disease may be either congenital or acquired.

The term “benign prostatic hyperplasia” refers to the increase in sizeof the prostate, for example, in middle-aged and elderly men.

As used herein, “crosslink,” “crosslinked” and “crosslinking” generallyrefer to the linking of two or more stabilizing materials, includinglipid, protein, polymer, carbohydrate, surfactant stabilizing materials,bioactive therapeutic factor and/or bioactive agents, by one or morebridges. The bridges may be composed of one or more elements, groups, orcompounds, and generally serve to join an atom from a first stabilizingmaterial molecule to an atom of a second stabilizing material molecule.The crosslink bridges may involve covalent and/or noncovalentassociations. Any of a variety of elements, groups, and/or compounds mayform the bridges in the crosslinks, and the stabilizing materials may becrosslinked naturally or through synthetic means. For example,crosslinking may occur in nature in material formulated from peptidechains which are joined by disulfide bonds of cystine residues, as inkeratins, insulins and other proteins. Alternatively, crosslinking maybe effected by suitable chemical modification, such as, for example, bycombining a compound, such as a stabilizing material, and a chemicalsubstance that may serve as a crosslinking agent, which may cause toreact by, for example, exposure to heat, high-energy radiation, and thelike. Examples include crosslinking by sulfur to form disulfidelinkages, crosslinking using organic peroxides, crosslinking ofunsaturated materials by means of high-energy radiation, crosslinkingwith dimethylol carbamate, and the like. In certain embodiments of themicrospheres provided herein, the gelatin is crosslinked.

The term “cell adhesion promoter” as used herein means any materialthat, because of their presence in or association with the microspheres,promotes or enhances the adhesiveness of cells to the surface of themicrospheres. These materials are often proteins that are bound to thesurface of the microspheres through covalent bonds of the proteins andthe polymers.

As used herein, “chemical modification” refers to the changes ofchemical properties and characteristics of the microspheres, eitherduring their production process or by way of mixing or contacting themwith various agents or tissues, such that the microspheres have theability to perform, in addition to embolization, other functions, forexample, once injected into the body.

The term “effective amount” as used herein refers to the amount of atherapy (e.g., a microsphere or composition provided herein) which issufficient to partially or completely occlude a blood vessel, such as anartery or vein. Such occlusion may be temporary or permanent. In certainembodiments, the effective amount will further ameliorate the severityand/or duration of a given disease and/or a symptom related thereto. Incertain embodiments of the methods provided herein, an effective amountof the microspheres is administered to the patient.

The term “embolization” or “therapeutic embolization” as used hereinrefers to a partial or total occlusion of vessels where the blood isflushing, for example, selective occlusion of blood vessels by purposelyintroducing emboli into the vessels. For example, embolization allowsocclusion of arteries or veins either to correct a dysfunction (e.g., anarteriovenous malformation) or to stop or slow the blood flow (e.g., toa solid tumor/cancer growth). In certain embodiments, embolization is apassive operation in the sense that no active molecules are carriedand/or delivered where the embolic material is deposited.

The term “in combination” as used herein in the context of theadministration of other therapies refers to the use of more than onetherapy. The use of the term “in combination” does not restrict theorder in which therapies are administered to a subject. A first therapycan be administered before (e.g., 1 minute, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) the administration of asecond therapy to a subject, for example, which had, has, or issusceptible to a given disease. Any additional therapy can beadministered in any order with the other additional therapies. Incertain embodiments, the particles provided herein can be administeredin combination with one or more therapies (e.g., therapies that are notmicrospheres that are currently administered to prevent, treat, manage,and/or ameliorate a given disease or other symptom related thereto).Non-limiting examples of therapies that can be administered incombination with the particles provided herein include analgesic agents,anesthetic agents, antibiotics, or immunomodulatory agents or any otheragent listed in the U.S. Pharmacopoeia—National Formulary (2009) U.S.Pharmacopoeia, including revisions, and/or Physician's Desk Reference(2009) 63^(rd) ed., Thomson Reuters.

As used herein, the term “impurity” or “impurities” refers to substancespresent in a confined amount of material or compound (e.g., a monomer ormonomer unit), which differ from the chemical composition of thematerial or compound. Impurities can be naturally occurring or addedduring or resulting from the synthesis of a material or compound.

As used herein, “injectable” means capable of being administered,delivered or carried into the body via syringe, catheters, needles orother means for injecting or infusing the microspheres in a liquidmedium. In certain embodiments, the particles provided herein areinjectable particles.

As used herein, a liquid or solution that has “low miscibility in water”refers to a liquid or solution having about 50% or less, about 40% orless, about 35% or less, about 30% or less, about 25% or less, about 20%or less, about 15% or less, about 10% or less, about 5% or less, about4% or less, about 3% or less, about 2% or less, about 1% or less, about0.5%, about 0.1% or less, or about 0% miscibility in water at 25° C. Ina specific embodiment, the liquid or solution has about 5% or lessmiscibility in water at 25° C. In another specific embodiment, theliquid or solution has about 1% or less miscibility in water at 25° C.In some embodiments, the liquid or solution is immiscible.

As used herein, the terms “manage,” “managing,” and “management” referto the beneficial effects that a subject derives from a therapy (e.g.,microspheres provided herein), which does not result in a cure of thedisease. In certain embodiments of the methods provided herein, asubject is administered one or more therapies to “manage” a givendisease or one or more symptoms related thereto, so as to prevent theprogression or worsening of the disease.

As used herein, the term “microspheres” refers to a polymer or one ormore combinations of polymers made into bodies of various sizes. Themicrospheres as used herein can be in any shape. In certain embodiments,the microspheres are substantially spherical shape. These structures ofthe microspheres may be generally spherical or spheroid in shape orbounded by imaginary spherical or spheroid shapes. In some embodiments,the surfaces of the microspheres provided herein appear smooth undermagnification of up to 1000 times, such as up to 100 times, up to 10times, 0 times or a range thereof. The microspheres may be sterilized byany method known in the art, for example, by irradiation, such as gammaor beta irradiation. The microspheres provided herein may comprise othermaterials as described and defined herein. However, it will beappreciated, that the term “microsphere” represents a convenientdescription for the purposes of explanation of the compositions andmethods provided herein, and that, in certain embodiments, the exemplarymicrospheres described herein are not necessarily limited to beingprecisely spherical in shape (e.g., are particles).

The term “monomer” refers to a small molecule that may become chemicallybonded to other monomers to form a polymer or copolymer. Reference tocertain properties or characteristics of a monomer herein can also applyto the corresponding monomer unit and vice versa.

The term “monomer unit” refers to a monomer in a polymer or copolymer.

The term “pharmaceutically acceptable” as used herein means beingapproved by a regulatory agency of the Federal or a state government, orlisted in the U.S. Pharmacopeia, European Pharmacopeia or othergenerally recognized pharmacopeia for use in animals, and moreparticularly in humans.

As used herein, the terms “prevent,” “preventing,” and “prevention”refer to the total or partial inhibition of a given disease; the totalor partial inhibition of the development or onset of disease progressionof given disease, or a symptom related thereto in a subject.

As used herein, the term “side effects” encompasses unwanted and adverseeffects of a therapy. Unwanted effects are not necessarily adverse. Anadverse effect from a therapy might be harmful or uncomfortable orrisky. Examples of side effects include, but are not limited to,rhinitis, diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting,anorexia, abdominal cramping, fever, pain, loss of body weight,dehydration, alopecia, dyspenea, insomnia, dizziness, mucositis, nerveand muscle effects, fatigue, dry mouth, and loss of appetite, rashes orswellings at the site of administration, flu-like symptoms such asfever, chills and fatigue, digestive tract problems and allergicreactions. Additional undesired effects experienced by patients arenumerous and known in the art. Many are described in the Physician'sDesk Reference (58th ed., 2004).

The term “sonication” refers to the act of applying sound energy (e.g.,ultrasonication) to agitate particles in a sample (e.g., microspheres).

As used herein, “stabilizing material” or “stabilizing compound” refersto any material which is capable of improving the stability ofcompositions, targeting ligands and/or other bioactive therapeuticfactors described herein, including, for example, mixtures, suspensions,emulsions, dispersions, vesicles, or the like.

The term “stem cells” refers to cells that have the capacity toself-renew and to generate differentiated progeny. In certainembodiments, the stem cells are mesenchymal stem cells.

The term “sticking” or “aggregated” refers to the state where an objector article (e.g., a microsphere) is in tight physical contact with oneor more objects or articles and is inseparable from other objects orarticles without external forces (e.g., gathering into a mass or whole).In certain embodiments, a sticking or aggregated microsphere refers to amicrosphere that is in tight physical contact with one or moremicrospheres, for example, due to the gelatin or a gelatin substitute.The term “unsticking,” “non-sticking,” “unaggregated” or“non-aggregated” refers to the state free from tight physical contact oradhesion. In certain embodiments, an unsticking, non-sticking,unaggregated or non-aggregated microsphere refers to a microsphere thatis substantially free or completely free from tight physical contactwith other microspheres. The percentage of sticking microspheres in apopulation can be determined, for example, by observation under amicroscope.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is a mammal such as anon-primate (e.g., cows, pigs, horses, cats, dogs, rats, rabbits, etc.)or a primate (e.g., monkey and human) comprising administration ofparticles as provided herein. In some embodiments, the patient is inneed of treatment or management of the disease or symptom thereof. Inspecific embodiments, the subject is a human.

As used herein, the term “substantially spherical” or “generallyspherical” refers to a shape that is close to a perfect sphere, which isdefined as a volume that presents the lowest external surface area.Specifically, “substantially spherical” as used herein means, whenviewing any cross-section of the particle (e.g., under a microscope),the difference between the average major diameter and the average minordiameter is less than 20%, less than 15%, less than 10%, less than 5%,or less than 1%. In some embodiments, the surfaces of the microspheresprovided herein appear smooth under magnification of up to 1000 times,such as up to 100 times, up to 10 times, 0 times or a range thereof.

The terms “therapeutic agent” or “therapeutic drug” can be usedinterchangeably herein and refers to any therapeutically activesubstance that is delivered to a bodily conduit of a living being toproduce a desired, usually beneficial, effect.

As used herein, the term “therapy” refers to any protocol, method and/oragent that can be used in the management, treatment and/or ameliorationof a given disease, or a symptom related thereto. In certainembodiments, the terms “therapies” and “therapy” refer to a biologicaltherapy, supportive therapy, and/or other therapies known to one ofskill in the art, such as medical personnel, useful in the management ortreatment of a given disease, or symptom related thereto.

As used herein, the terms “treat,” “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity, and/orduration of a disease or a symptom thereof.

As used herein, the term “ultra-pure” refers to the state of a compoundor material (e.g., monomer or monomer unit) having a very low level ofimpurities compared to a compound or material (e.g., monomer or monomerunit) that is undiluted or unmixed with extraneous compounds ormaterials. In certain embodiments, the impurity is a salt. In someembodiments, the level of impurities present in a compound or materialcan be expressed as a percentage (%); for example, an ultra-pure monomeror an ultra-pure monomer unit can have less than 1%, less than 2%, lessthan 3%, less than 4%, less than 5%, less than 6%, less than 7%, lessthan 8%, less than 9%, less than 10%, less than 15%, less than 20%, lessthan 25% of impurities or any range thereof. In certain embodiments, thelevel of impurities is determined by the bromine test (see, e.g.,Examples 3 and 6). In other embodiments, the level of impurities isdetermined by HPLC (see e.g., Examples 2 and 5).

The term “uterine fibroid” or “leiomyoma” refers to non-cancerous tumorscomposed of certain types of muscle fibers and fibrous connective tissueof the uterus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate a comparison of the purity of monomers. FIG. 1Adepicts the differences in the sensitivities of purity observed using abromination reaction (right bars, as provided by manufacturer BioSepra)and HPLC (left bars) for given lots of DEAE monomer (T209, U088 andU089) obtained from BioSepra. FIG. 1B depicts the difference in thesensitivities of purity for two different lots of trisacryl (R426 andR402 obtained from BioSepra) observed using a bromination reaction(right bars) or HPLC (left bars), and the data are shown with anassumption of 100% purity of a recrystallized trisacryl (TA-R) obtainedand compared with trisacryl monomer from SAFC prior to recrystallization(TA).

FIGS. 2A-2C illustrate the reduction of sticking microspheres byultrasonication. The image analyses shows a significant decrease ofsticking microspheres by ultrasonication. No broken microspheres wereobserved by this method. FIG. 2A shows microspheres before crosslinking,initially (left panel) and after 15 min of ultrasonication (rightpanel). The percentage of sticking microspheres decreases from about 7%to about 0.2% with ultrasonication. FIG. 2B demonstrates microspheresafter crosslinking without (left panel) or with (right panel)ultrasonication prior to crosslinking. Sieving was done after theultrasonication step. The percentage of sticking microspheres decreasesfrom about 3% to nearly about 0% with ultrasonication. FIG. 2C showsmicrospheres before crosslinking, initially (left panel) and after 2×15min. of ultrasonication (right panel). The percentage of stickingmicrospheres decreases from about 9.2% to about 1.6% withultrasonication.

FIGS. 3A-3C illustrate the percentages of sticking microspheres subjectto ultrasonication before (FIG. 3A) and after gelatin crosslinking (FIG.3B). Sieving was made after the ultrasonication step, and the percentageof volume on sieve is shown in FIG. 3C.

FIGS. 4A-4G illustrate the high resolution magic angle spinning (HR-MAS)one-dimensional (ID) ¹H spectra of starting materials and microspheres.FIGS. 4A-D illustrate the HR-MAS one-dimensional ¹H spectra of trisacryl(4A), gelatin (4B), MBA (4C) and DEAE acrylamide (4D). FIG. 4E-4G showthe attribution of 1H nuclei of trisacryl (4E), MBA (4F) and DEAEacrylamide (4G).

FIGS. 5A-5J illustrate the one-dimensional ¹H NMR spectra ofmicrospheres made from materials of different sources. FIGS. 5A-5Dillustrate the NMR spectra of samples SAFC FMP 128 and PALL FMP 130.FIG. 5A is the superposition of the NMR spectra showing a similaritybetween the corresponding main peaks (marked as A, B and C) identifiedfrom the spectra. FIG. 5B shows the NMR spectra in the region of 9-5ppm. FIG. 5C shows the NMR spectra in the region of 5-0 ppm. FIG. 5Dshows the NMR spectra in the region of 3.4-1.2 ppm. FIGS. 5E-5Jillustrate the NMR spectra of samples SAFC FMP 128, PALL FMP 130,FM0903031-M1675 and FM0903021-M1654. FIG. 5E shows a comparison of theNMR spectra. FIG. 5F shows the NMR spectra of 2.6-1.5 ppm. FIG. 5G showsthe spectra in the region of 3.4-1.2 ppm. FIG. 5H shows the NMR spectrain the region of 9-5 ppm. FIG. 5I shows a superposition of the spectra(FIG. 5I). FIG. 5J shows the NMR spectra after deconvolution.

FIG. 6 illustrates a schematic diagram of an illustrative Feed Ringprocess.

FIG. 7 illustrates a HR-MAS rotor used for the NMR analysis.

DETAILED DESCRIPTION

The microspheres provided herein are, in certain embodiments, non-toxicto organs, tissues and cells, biocompatible, and adhesive to variouscells and tissues at the site of implantation by means of the cellgrowth they promote. In addition, in certain embodiments, thesemicrospheres are non-resorbable and non-biodegradable, and thus arestable, durable, and will maintain their general shape and position onceimplanted at a desired site. In further embodiments, the microspheresare also stable in suspension which allows the microspheres or othersolid substrates to be formulated and stored in suspension and injectedwith different liquids.

A. Microspheres for Embolization

In one aspect, provided herein is a microsphere comprising: (a) acopolymer comprising a N-tris-hydroxymethyl methylacrylamide monomerunit, a diethylaminoethylacrylamide monomer unit and aN,N-methylene-bis-acrylamide monomer unit, and (b) a crosslinked gelatinor gelatin substitute. In another aspect, provided herein is amicrosphere comprising (a) a copolymer prepared by copolymerizing aN-tris-hydroxymethyl methylacrylamide monomer, adiethylaminoethylacrylamide monomer and a N,N-methylene-bis-acrylamidemonomer, and (b) a crosslinked gelatin or gelatin substitute. Inspecific embodiments, one, two or all three of the N-tris-hydroxymethylmethylacrylamide, diethylaminoethylacrylamide and aN,N-methylene-bis-acrylamide monomers (or monomer units) are ultra-puremonomers (or monomer units). In certain embodiments, the microsphereexhibits in a ¹H NMR spectrum, a first peak (e.g., from about 3.5 ppm toabout 4 ppm), a second peak (e.g., from about 3 ppm to about 3.5 ppm),and a third peak (e.g., from about 1 ppm to about 1.5 ppm). In specificembodiments, the integration ratio of the second peak to the first peakis from about 0.50 to about 0.65 and/or the integration ratio of thethird peak to the first peak is from about 0.55 to about 0.75 (e.g.,about 0.61 to about 0.75).

In one embodiment, the microsphere comprises: (a) a copolymer comprisinga N-tris-hydroxymethyl methylacrylamide monomer unit, adiethylaminoethylacrylamide monomer unit and aN,N-methylene-bis-acrylamide monomer unit, and (b) crosslinked gelatin;wherein the microsphere exhibits in a ¹H NMR spectrum, a first peak fromabout 3.5 ppm to about 4 ppm, a second peak from about 3 ppm to about3.5 ppm, and a third peak from about 1 ppm to about 1.5 ppm; and wherein(i) the integration ratio of the second peak to the first peak is about0.50 to about 0.65, (ii) the integration ratio of the third peak to thefirst peak is about 0.55 to about 0.75 (e.g., about 0.61 to about 0.75),or (iii) a combination of (i) and (ii). In another embodiment, themicrosphere comprises (a) a copolymer prepared by copolymerizing aN-tris-hydroxymethyl methylacrylamide monomer, adiethylaminoethylacrylamide monomer and a N,N-methylene-bis-acrylamidemonomer, and (b) crosslinked gelatin; wherein the microsphere exhibitsin a ¹H NMR spectrum, a first peak from about 3.5 ppm to about 4 ppm, asecond peak from about 3 ppm to about 3.5 ppm, and a third peak fromabout 1 ppm to about 1.5 ppm; and wherein (i) the integration ratio ofthe second peak to the first peak is about 0.50 to about 0.65, (ii) theintegration ratio of the third peak to the first peak is about 0.55 toabout 0.75 (e.g., about 0.61 to about 0.75), or (iii) a combination of(i) and (ii). In certain embodiments, the first peak is at about 3.77ppm, the second peak is at about 3.2 ppm, the third peak is at about 1.3ppm, or a combination thereof. In other embodiments, the integrationratio of the second peak to the first peak is about 0.574, or whereinthe integration ratio of the third peak to the first peak is about0.625. In some embodiments, the microsphere is at 25° C. and/or in adeuterated solvent when the ¹H NMR spectrum is recorded (e.g., at 400MHz).

In some embodiments, one or more of the monomers (or monomer units) isan ultra-pure monomer (or monomer units). In certain embodiments, theN-tris-hydroxymethyl methylacrylamide monomer, thediethylaminoethylacrylamide monomer and/or theN,N-methylene-bis-acrylamide monomer are each ultra-pure monomers. Inspecific embodiments, the N-tris-hydroxymethyl methylacrylamide monomercomprises less than 9% of impurities and/or thediethylaminoethylacrylamide monomer comprises less than 2% of impurities(e.g., as determined by HPLC (see, e.g., Examples 2 and 5) or by abromine test (see, e.g., Examples 3 and 6). In certain embodiments, theN-tris-hydroxymethyl methylacrylamide monomer, thediethylaminoethylacrylamide monomer and the N,N-methylene-bis-acrylamidemonomer are each ultra-pure monomers. In one embodiment, theN-tris-hydroxymethyl methylacrylamide monomer is an ultra-pure monomer,while the diethylaminoethylacrylamide monomer andN,N-methylene-bis-acrylamide monomer are not ultra-pure monomers. Inanother embodiment, the N-tris-hydroxymethyl methylacrylamide monomerand the diethylaminoethylacrylamide monomer are each ultra-puremonomers, while the N,N-methylene-bis-acrylamide monomer is not anultra-pure monomer. In some embodiments, the N-tris-hydroxymethylmethylacrylamide monomer and the N,N-methylene-bis-acrylamide monomerare each ultra-pure monomers, while the diethylaminoethylacrylamidemonomer is not an ultra-pure monomer. In one embodiment, thediethylaminoethylacrylamide monomer is an ultra-pure monomer, while theN-tris-hydroxymethyl methylacrylamide monomer and theN,N-methylene-bis-acrylamide monomer are not ultra-pure monomers. Inanother embodiment, the diethylaminoethylacrylamide monomer and theN,N-methylene-bis-acrylamide monomer are each ultra-pure monomers, whilethe N-tris-hydroxymethyl methylacrylamide is not an ultra-pure monomer.In one embodiment, the N,N-methylene-bis-acrylamide monomer is anultra-pure monomer, while the N-tris-hydroxymethyl methylacrylamidemonomer and the diethylaminoethylacrylamide monomer are not ultra-puremonomers. In another embodiment, the diethylaminoethylacrylamide monomeris an ultra-pure monomer while the N-tris-hydroxymethyl methylacrylamidemonomer is not. In yet another embodiment, both the N-tris-hydroxymethylmethylacrylamide and diethylaminoethylacrylamide monomers are ultra-puremonomers. In further embodiments, the hydrophobicity or ionic characterof these monomers can be modified as deemed necessary by introducing forexample, without limitation, hydrocarbon chains and/or hydrophilicionizable chemical groups, which can, for example, be used to facilitatedrug-loading characteristics of the microsphere (e.g., ionicinteractions between the copolymer and the drug). For example, incertain embodiments, the monomer or polymer is modified to add acidicfunctional groups (e.g., addition of sodium acrylate or vinyl sulfonateto monomer mixture), which can, for example interact with aminefunctions in a given drug (e.g., doxorubicin or other anthracyclines).In a specific embodiment, monomer is modified with sulfonate groups. theIn some embodiments, the monomer has a moisture content of from about 5%to about 0%, such as 5% or less, 4% or less, 3% or less, 2% or less or1% or less. Moisture content can be determined using methods known inthe art, such as NMR analysis.

In some embodiments, the gelatin is crosslinked, for example, to orwithin the copolymer of the microsphere. In other embodiments, thegelatin is not crosslinked. The gelatin as used herein can be from anysource. Exemplary sources include, but are not limited to, collagensextracted from bones, connective tissues, organs and some intestines ofcattle, pigs, and horses. In a specific embodiment, the gelatin is aporcine gelatin. In specific embodiments, the gelatin is pharmaceuticaland/or food grade gelatin, which can be obtained from commercialsuppliers, such as PB Leiner (Vilvoorde, Belgium).

In a second aspect, provided herein are microspheres prepared by aprocess comprising: (a) preparing an aqueous solution comprising (i) aN-tris-hydroxymethyl methylacrylamide monomer, (ii) adiethylaminoethylacrylamide monomer, (iii) aN,N-methylene-bis-acrylamide monomer, and (iv) gelatin, wherein theN-tris-hydroxymethyl methylacrylamide monomer, thediethylaminoethylacrylamide monomer and/or theN,N-methylene-bis-acrylamide monomer is an ultra-pure monomer; (b)adding the aqueous solution to a liquid organic phase (e.g., an oil,such as a mineral oil, such as a paraffin oil) that has low miscibilityin water, before or while stirring; thereby producing microspherescomprising a copolymer comprising N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and N,N-methylene-bis-acrylamide monomerunits; (c) optionally subjecting the microspheres to sonication (e.g.,ultrasonication); and (d) crosslinking the gelatin. Embodiments of theprocess are described in Section C below.

Microspheres provided herein can have any shape. In specificembodiments, these microspheres are substantially spherical in shape. Incertain embodiments, the microspheres are uniform shape.

In certain embodiments, the microspheres provided are calibrated to acertain size range. Such calibration can be achieved using method knownin the art, such as by one or more rounds of sieving using anappropriately sized mesh sieve.

In certain embodiments, the microspheres provided herein have a diameterfrom about 1 μm to 2000 μm, such as from about 10 μm to 1000 μm, fromabout 40 μm to about 120 μm, from about 100 μm to about 300 μm, fromabout 300 μm to about 500 μm, from about 500 μm to about 700 μm, fromabout 700 μm to about 900 μm, or from about 900 μm to about 1200 μm.These diameters can permit the microspheres to be delivered to targetblood vessels, tissues or organs in vivo via catheter, needle (e.g., a18 gauge or smaller needle), tubing, or the like by various pathwaysincluding vascular, intraductal, transesophogeal, subcutaneous,subdermal, submucosal, transbronchial, or interstitial. In certainembodiments, the microspheres can be eliminated through macrophages orother elements of the immune system or the lymphatic system.

In certain embodiments, the microspheres are uniform in size. In certainembodiments, the microspheres are uniform in size, wherein thedifference in diameter between individual microspheres is from about 0μm to about 100 μm, from about 0 μm to about 50 μm, or from about 0 μmto about 25 μm, such as 100 μm or less, about 50 μm or less, about 25 mmor less, about 10 mm or less or about 5 μm or less.

In certain embodiments, the microspheres are in a population whereingreater than 68% have a diameter of ±20% of the mean diameter, ±10% ofthe mean diameter, ±15% of the mean diameter, ±10% of the mean diameter,±9% of the mean diameter, ±8% of the mean diameter, ±7% of the meandiameter, ±6% of the mean diameter, ±5% of the mean diameter, ±4% of themean diameter, ±3% of the mean diameter, ±2% of the mean diameter, or±1% of the mean diameter, or any range thereof. In one embodiment, themicrospheres are in a population wherein greater than 70% have adiameter of ±20% of the mean diameter, ±10% of the mean diameter, ±15%of the mean diameter, ±10% of the mean diameter, ±9% of the meandiameter, ±8% of the mean diameter, ±7% of the mean diameter, ±6% of themean diameter, ±5% of the mean diameter, ±4% of the mean diameter, ±3%of the mean diameter, ±2% of the mean diameter, or ±1% of the meandiameter, or any range thereof. In one embodiment, the microspheres arein a population wherein greater than 75% have a diameter of ±20% of themean diameter, ±10% of the mean diameter, ±15% of the mean diameter,±10% of the mean diameter, ±9% of the mean diameter, ±8% of the meandiameter, ±7% of the mean diameter, ±6% of the mean diameter, ±5% of themean diameter, ±4% of the mean diameter, ±3% of the mean diameter, ±2%of the mean diameter, or ±1% of the mean diameter, or any range thereof.In one embodiment, the microspheres are in a population wherein greaterthan 80% have a diameter of ±20% of the mean diameter, ±10% of the meandiameter, ±15% of the mean diameter, ±10% of the mean diameter, ±9% ofthe mean diameter, ±8% of the mean diameter, ±7% of the mean diameter,±6% of the mean diameter, ±5% of the mean diameter, ±4% of the meandiameter, ±3% of the mean diameter, ±2% of the mean diameter, or ±1% ofthe mean diameter, or any range thereof. In one embodiment, themicrospheres are in a population wherein greater than 85% have adiameter of ±20% of the mean diameter, ±10% of the mean diameter, ±15%of the mean diameter, ±10% of the mean diameter, ±9% of the meandiameter, ±8% of the mean diameter, ±7% of the mean diameter, ±6% of themean diameter, ±5% of the mean diameter, ±4% of the mean diameter, ±3%of the mean diameter, ±2% of the mean diameter, or ±1% of the meandiameter, or any range thereof. In one embodiment, the microspheres arein a population wherein greater than 90% have a diameter of ±20% of themean diameter, ±10% of the mean diameter, ±15% of the mean diameter,±10% of the mean diameter, ±9% of the mean diameter, ±8% of the meandiameter, ±7% of the mean diameter, ±6% of the mean diameter, ±5% of themean diameter, ±4% of the mean diameter, ±3% of the mean diameter, ±2%of the mean diameter, or ±1% of the mean diameter, or any range thereof.In one embodiment, the microspheres are in a population wherein greaterthan 95% have a diameter of ±20% of the mean diameter, ±10% of the meandiameter, ±15% of the mean diameter, ±10% of the mean diameter, ±9% ofthe mean diameter, ±8% of the mean diameter, ±7% of the mean diameter,±6% of the mean diameter, ±5% of the mean diameter, ±4% of the meandiameter, ±3% of the mean diameter, ±2% of the mean diameter, or ±1% ofthe mean diameter, or any range thereof.

In some embodiments, less than 1%, less than 0.9%, less than 0.8%, lessthan 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than0.3% or less than 0.2%, less than 0.1% or 0% (or a range thereof) ofmicrospheres in a population are outside of a given extended range(e.g., ±50 μm±100 μm, ±150 μm, ±200 μm, ±250 μm or ±300 μm) of themicrospheres. As an exemplary illustration, if the diameter of apopulation of calibrated microspheres is from 500 μm to 700 μm (therange), then the extended range can be, for example, ±100 μm, e.g.,wherein 99% of the microspheres are of a size range of from 400 μm to800 μm, and 1% of the microspheres are of a size range outside theextended range. In another exemplary embodiment, the range is 500 μm to700 μm, with the nominal range being 600 μm, wherein 80% of thepopulation is ±100 μm of the nominal range (i.e., an extended range of500 μm to 700 μm), 99% of the population is ±200 μm of the nominal range(i.e., 400 μm to 800 μm), with 0.5% to 1% of the population being largerthan 800 μm and the remaining 0% to 0.5% being of the population smallerthan 400 μm (for a total of 100%). In certain embodiments, the nominalrange is from about 50 μm to 2000 μm, such as 80 μm, 100 μm, 200 μm, 300μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm,1150 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm,1900 μm, 2000 μm or any range thereof.

The microspheres are stable in suspension, which allows the microspheresto be formulated and stored in suspension and injected with differentliquids. More specifically, the hydrophilic nature of the microspherespermits placing them in suspension, and in particular, in the form ofsterile and non-pyrogenic or pyrogen-free injectable solutions, whileavoiding the formation of aggregates or adhesion to the walls of storagecontainers and implantation devices, such as catheters, syringes,needles, and the like.

Microspheres provided herein can be implanted, such as by injection, invarious locations of the body. The polymeric material for use in thecompositions and methods provided herein is non-toxic to tissues andcells and is biocompatible, i.e., generally does not cause inflammation.The microspheres can maintain their general shape and position onceimplanted at a desired site. The microspheres provided herein arecompressible and, in specific embodiments, can be injected throughneedles of 18 gauge or smaller.

In certain embodiments, the microspheres are injectable through a needleof 18 gauge or smaller and are not capable of being eliminated, or havereduced elimination, by the immune or lymphatic system. In someembodiments, the polymers are coated with agents which promote celladhesion. In certain embodiments, the microspheres comprise a celladhesion promoter in addition to the gelatin or gelatin substitute.Various types of cell adhesion promoters well known in the art can beused. In some embodiments, the cell adhesion promoter is selected fromcollagen, gelatin, carboxymethyl (CM) dextran, DEAE dextran,glucosaminoglycans, fibronectin, lectins, polycations (such aspolylysine, chitosan), any other natural or synthetic biological celladhesion agent or any combinations thereof. In certain embodiments, thestability of the microspheres is increased by reticulating the adhesionagent. In the case of gelatin, for example, the reticulating agent canbe chosen among the difunctional chemical agents reacting on the gelatinamines (e.g., glutaraldehyde, formaldehyde, glyoxal, and the like). Insome embodiments, the cell adhesion promoter is present in themicrosphere, or other solid substrate, in an amount from about 0.1 g/mlto 1 g/ml of settled microspheres.

In certain embodiments, the microspheres are visible in the light andwithin the body by, for example, further comprising a marking agent. Insome embodiment, microspheres can be marked after their synthesis. Thiscan be done, for example, by grafting of fluorescent markers derivatives(including, for example, fluorescein isothiocyanate (FITC), rhodamineisothiocyanate (RITC) and the like). In some embodiments, a detectablemonomer can be obtained by chemical coupling of the monomer with amarker, which can be: a chemical dye, such as Cibacron Blue or ProcionRed HE-3B, making possible a direct visualization of the microspheres, amagnetic resonance imaging agent (erbium, gadolinium or magnetite); acontrasting agent, such as barium or iodine salts, including, forexample, (acrylamido-3-propionamido)-3-triiodo-2,4,6-benzoic acid. Inthe case of barium or magnetite salts, they can be directly introducedin powered form in the initial monomer solution. In a certainembodiment, the contrast agent is a radiopaque contrast agent, such as anon-ionic contrast agent. In some embodiments, the contrast agent ismixed with the microsphere prior to, during and/or after injection intothe patient. In a specific embodiment, the patient is administered acomposition comprising a contrast agent (e.g., a non-ionic contrastagent) and microspheres provided herein.

Cell adhesion promoters or marking agents can be introduced onmicrospheres by chemical coupling procedures well known in affinitychromatography. The introduction can also be accomplished by diffusionwithin the gel network that constitutes the microsphere and thentrapping the diffused molecules in place by precipitation or chemicalcrosslinking. In some embodiments, living cells (e.g., stem cells) areattached to the microspheres forming layers of cells therein or thereonthat link with surrounding tissues and can enhance the long-termstability of the beads.

1. N-tris-hydroxymethyl methylacrylamide Monomer

N-tris-hydroxymethyl methylacrylamide can also be known as trisacryl,N-[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]prop-2-enamide;TRIS-acrylamide; N-[tris(hydroxymethyl)methyl]acrylamide;N-acryloyltris(hydroxymethyl)aminomethane; orN-acryloyl-tris(hydroxymethyl)aminomethane. In certain embodiments,N-tris-hydroxymethyl methylacrylamide is defined as having a CASRegistry Number 13880-05-2, a molecular formula of C₇H₁₃NO₄, a molecularweight of about 175.2 grams per mole, and a structure of:

N-tris-hydroxymethyl methylacrylamide monomers are commerciallyavailable (e.g., PALL Biosepra, France or Sigma Aldrich Fine Chemicals(SAFC), product number 78561) or can be synthesized (see, e.g., Example4). In certain embodiments, the N-tris-hydroxymethyl methylacrylamidemonomer is synthesized according to the following scheme:

In certain embodiments, the N-tris-hydroxymethyl methylacrylamidemonomer is an ultra-pure monomer. In specific embodiments, theultra-pure N-tris-hydroxymethyl methylacrylamide monomer comprises from0% to 9% of impurities, i.e., substances other than N-tris-hydroxymethylmethylacrylamide such as excess starting materials or derivativesthereof (e.g., acryloyl chloride or2-amino-2-(hydroxymethyl)propane-1,3-diol), by-products or inorganicsalts, for example, as determined by the bromine test (e.g., as providedin Example 6). In some embodiments, the N-tris-hydroxymethylmethylacrylamide monomer comprises from 25% to 0% impurities, such asfrom 25% to 20%, from 25% to 15%, from 25% to 10%, from 25% to 5%, from25% to 1%, from 25% to 0%, from 20% to 15%, from 20% to 10%, from 20% to5%, from 20% to 1%, from 20% to 0%, from 15% to 10%, from 15% to 5%,from 15% to 1%, from 15% to 0%, from 10% to 5%, from 10% to 1%, from 10%to 0%, from 5% to 1%, from 5% to 0% (e.g., as determined by HPLC, e.g.,as provided in Example 5). In other embodiments, theN-tris-hydroxymethyl methylacrylamide monomer comprises from 25% to 0%impurities, such as from 25% to 20%, from 25% to 15%, from 25% to 10%,from 25% to 5%, from 25% to 1%, from 25% to 0%, from 20% to 15%, from20% to 10%, from 20% to 5%, from 20% to 1%, from 20% to 0%, from 15% to10%, from 15% to 5%, from 15% to 1%, from 15% to 0%, from 10% to 5%,from 10% to 1%, from 10% to 0%, from 5% to 1%, from 5% to 0% (e.g., asdetermined by HPLC, e.g., as provided in Example 5). In someembodiments, the N-tris-hydroxymethyl methylacrylamide monomer comprisesless than 25%, less than 24%, less than 23%, less than 22%, less than21%, less than 20%, less than 9%, less than 18%, less than 17%, lessthan 16%, less than 15%, less than 14%, less than 13%, less than 12%,less than 11%, less than 10%, less than 9%, less than 8%, less than 7%,less than 6%, less than 5%, less than 4%, less than 3%, less than 2.75%,less than 2.5%, less than 2.25%, less than 2%, less than 1.75%, lessthan 1.5%, less than 1.25%, less than 1%, less than 0.25%, less than0.5%, less than 0.25% or 0% of impurities or any range thereof (e.g., asdetermined by the bromine test, e.g., as provided in Example 6). Inother embodiments, the N-tris-hydroxymethyl methylacrylamide monomercomprises less than 25%, less than 24%, less than 23%, less than 22%,less than 21%, less than 20%, less than 9%, less than 18%, less than17%, less than 16%, less than 15%, less than 14%, less than 13%, lessthan 12%, less than 11%, less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2.75%, less than 2.5%, less than 2.25%, less than 2%, less than1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.25%,less than 0.5%, less than 0.25% or 0% of impurities or any range thereof(e.g., as determined by HPLC, e.g., as provided in Example 5). Inspecific embodiments, the N-tris-hydroxymethyl methylacrylamide monomercomprises less than 9%, less than 8%, less than 7%, less than 6%, lessthan 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0%of impurities, or any range thereof (e.g., as determined by the brominetest, e.g., as provided in Example 6). In other embodiments, theN-tris-hydroxymethyl methylacrylamide monomer comprises less than 9%,less than 8%, less than 7%, less than 6%, less than 5%, less than 4%,less than 3%, less than 2%, less than 1%, or 0% of impurities, or anyrange thereof (e.g., as determined by HPLC, e.g., as provided in Example5).

2. Diethylaminoethylacrylamide Monomer

Diethylaminoethylacrylamide can also be known as DEAE, DEAE acrylamide,N-((2-diethylamino)ethyl)acrylamide;N-(2-diethylaminoethyl)prop-2-enamide;N-((2-diethylamino)ethyl)acrylamide;N-(2-(diethylamino)ethyl)acrylamide; orN-(2-(diethylamino)ethyl)-2-propenamide.

In certain embodiments, diethylaminoethylacrylamide is defined as havinga molecular formula of C₉H₁₈N₂O, a molecular weight of about 170.3 gramsper mole, and a structure of:

Diethylaminoethylacrylamide monomers are commercially available (e.g.,PALL Biosepra, France or SAFC) or can be synthesized (see, e.g., Example1). In certain embodiments, the diethylaminoethylacrylamide monomer issynthesized according to the following scheme:

In certain embodiments, the diethylaminoethylacrylamide monomer is anultra-pure monomer. In specific embodiments, the ultra-purediethylaminoethylacrylamide monomer, comprises from 0% to 2% ofimpurities, i.e., substances other than diethylaminoethylacrylamide suchas excess starting materials or derivatives thereof (e.g., acryloylchloride, N,N,N′,N′-tetramethylethylenediamine orN,N-diethylethylenediamine), by-products or inorganic salts (asdetermined by the bromine test, e.g., as provided in Example 3). In someembodiments, the diethylaminoethylacrylamide monomer comprises from 20%to 0% impurities, such as from 20% to 15%, from 20% to 10%, from 20% to5%, from 20% to 1%, from 20% to 0%, from 15% to 10%, from 15% to 5%,from 15% to 1%, from 15% to 0%, from 10% to 5%, from 10% to 1%, from 10%to 0%, from 5% to 1%, from 5% to 0% (e.g., as determined by the brominetest, e.g., as provided in Example 3). In other embodiments, thediethylaminoethylacrylamide monomer comprises from 20% to 0% impurities,such as from 20% to 15%, from 20% to 10%, from 20% to 5%, from 20% to1%, from 20% to 0%, from 15% to 10%, from 15% to 5%, from 15% to 1%,from 15% to 0%, from 10% to 5%, from 10% to 1%, from 10% to 0%, from 5%to 1%, from 5% to 0% (e.g., as determined by HPLC, e.g., as provided inExample 2). In some embodiments, the diethylaminoethylacrylamide monomercomprises less than 20%, less than 19%, less than 18%, less than 17%,less than 16%, less than 15%, less than 14%, less than 13%, less than12%, less than 11%, less than 10%, less than 9%, less than 8%, less than7%, less than 6%, less than 5%, less than 4%, less than 3%, less than2.75%, less than 2.5%, less than 2.25%, less than 2%, less than 1.75%,less than 1.5%, less than 1.25%, less than 1%, less than 0.25%, lessthan 0.5%, less than 0.25% or 0% of impurities or any range thereof(e.g., as determined by the bromine test, e.g., as provided in Example3). In other embodiments, the diethylaminoethylacrylamide monomercomprises less than 20%, less than 19%, less than 18%, less than 17%,less than 16%, less than 15%, less than 14%, less than 13%, less than12%, less than 11%, less than 10%, less than 9%, less than 8%, less than7%, less than 6%, less than 5%, less than 4%, less than 3%, less than2.75%, less than 2.5%, less than 2.25%, less than 2%, less than 1.75%,less than 1.5%, less than 1.25%, less than 1%, less than 0.25%, lessthan 0.5%, less than 0.25% or 0% of impurities or any range thereof(e.g., as determined by HPLC, e.g., as provided in Example 2). Inspecific embodiments, the DEAE monomer comprises less than 5%, less than4%, less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, lessthan 2%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%,less than 0.25%, less than 0.5%, less than 0.25% or 0% of impurities orany range thereof (e.g., as determined by the bromine test, e.g., asprovided in Example 3). In other specific embodiments, the DEAE monomercomprises less than 5%, less than 4%, less than 3%, less than 2.75%,less than 2.5%, less than 2.25%, less than 2%, less than 1.75%, lessthan 1.5%, less than 1.25%, less than 1%, less than 0.25%, less than0.5%, less than 0.25% or 0% of impurities or any range thereof (e.g., asdetermined by HPLC, e.g., as provided in Example 2). The DEAE monomercan be provided in a liquid or solid form. In certain embodiments, theDEAE monomer is provided in a liquid amine form. In specificembodiments, the DEAE monomer is not provided in a powder salt form(powder).

3. N,N-methylene-bis-acrylamide Monomer

N,N-methylene-bis-acrylamide can also be known as MBA,N-[(prop-2-enoylamino)methyl]prop-2-enamide; methylenediacrylamide;methylenebisacrylamide; N,N′-methylenediacrylamide;N,N′-methylenebisacrylamide; or N,N′-methylidenebisacrylamide.

In certain embodiments, N,N-methylene-bis-acrylamide is defined ashaving a molecular formula of C₇H₁₀N₂O₂, a molecular weight of about154.2 grams per mole, and a structure of:

N,N-methylene-bis-acrylamide monomers are commercially available (e.g.,PALL Biosepra, France or SAFC) or can be synthesized. In certainembodiments, the N,N-methylene-bis-acrylamide monomer is synthesizedaccording to the following scheme:

In certain embodiments, the N,N-methylene-bis-acrylamide monomer is anultra-pure monomer. In specific embodiments, the ultra-pureN,N-methylene-bis-acrylamide monomer comprises from 0% to 9% ofimpurities, i.e., substances other than N,N-methylene-bis-acrylamidemonomer such as excess starting materials or derivatives thereof,by-products or inorganic salts (e.g., as determined by the brominetest). In other embodiments, the ultra-pure N,N-methylene-bis-acrylamidemonomer comprises from 0% to 9% of impurities, i.e., substances otherthan N,N-methylene-bis-acrylamide monomer such as excess startingmaterials or derivatives thereof, by-products or inorganic salts (e.g.,as determined by HPLC). In some embodiments, theN,N-methylene-bis-acrylamide monomer comprises less than 9%, less than8%, less than 7%, less than 6%, less than 5%, less than 4%, less than3%, less than 2%, less than 1%, less than 0.5% or 0% of impurities, orany range thereof (e.g., as determined by the bromine test). In someembodiments, the N,N-methylene-bis-acrylamide monomer comprises lessthan 9%, less than 8%, less than 7%, less than 6%, less than 5%, lessthan 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or 0%of impurities, or any range thereof (e.g., as determined by HPLC).

The purity of any of the monomers provided herein can be assessed by avariety of methods known in the art. For example, the content ofimpurities can be determined by the content of double bonds in themonomers, for example, the bromine test (see, e.g., Examples 3 and 6).The bromine test can be used to determine if the compound contains anydouble C═C bonds, or the alkene functional group, because alkenes canreact readily with bromine to produce color change. Exemplarybromination reaction:

The bromine reacts with the double bond and the consumption of brominedetermines the quantity of double bond present, which is then used toevaluate so the purity, or the real presence of a monomer (e.g.,trisacryl). The standard deviation can be at least +/−3% using thisreaction. The quantity of double bonds present can correspond to thepurity of the monomer present, because side products should not bepresent in large quantities. Exemplary side products are:

The impurities present in the monomers can also be detected by othermethods with a higher detection sensitivity (e.g., high performanceliquid chromatography (HPLC), gas chromatography (GC) or nuclearmagnetic resonance (NMR)). In an illustrative example, an HPLC analysiscan be performed to assess the purity of N-tris-hydroxymethylmethylacrylamide monomers by running the monomer samples on a WatersAtlantic C₁₈ column (e.g., 5 μm, 4.6×250 mm) and detecting theabsorption at a specific wavelength, for example, at about 230 nm (see,e.g., Examples 2 and 5). The purity of the tested monomers can bedetermined, for example, by comparing the HPLC curve of the testedsamples to the HPLC curve of a control monomer with known purity.

In certain embodiments, the DEAE monomer comprises less than 2.5%, lessthan 2%, less than 1.5%, or less than 1% of each or both of thefollowing impurities (e.g., as determined by HPLC), either alone or incombination:

In some embodiments, the microsphere comprises about 1% to about 95% byweight of a N-tris-hydroxymethyl methylacrylamide monomer (e.g., anultra-pure monomer). In certain embodiments, the microsphere comprises aN-tris-hydroxymethyl methylacrylamide monomer (e.g., an ultra-puremonomer) in an amount selected from the group consisting of about 1% byweight, about 5% by weight, about 10% by weight, about 15% by weight,about 20% by weight, about 25% by weight, about 30% by weight, about 35%by weight, about 40% by weight, about 45% by weight, about 50% byweight, about 55% by weight, about 60% by weight, about 65% by weight,about 70% by weight, about 75% by weight, about 80% by weight, about 85%by weight, about 90% by weight, and about 95% by weight (or in rangethereof).

In certain embodiments, the microsphere comprises about 1% to about 95%by weight of a diethylaminoethylacrylamide monomer (e.g., an ultra-puremonomer). In certain embodiments, the microsphere comprises adiethylaminoethylacrylamide monomer (e.g., an ultra-pure monomer) in anamount selected from the group consisting of about 1% by weight, about5% by weight, about 10% by weight, about 15% by weight, about 20% byweight, about 25% by weight, about 30% by weight, about 35% by weight,about 40% by weight, about 45% by weight, about 50% by weight, about 55%by weight, about 60% by weight, about 65% by weight, about 70% byweight, about 75% by weight, about 80% by weight, about 85% by weight,about 90% by weight, and about 95% by weight (or in range thereof).

In other embodiments, the microsphere comprises about 1% to about 95% byweight of a N,N-methylene-bis-acrylamide monomer (e.g., an ultra-puremonomer). In certain embodiments, the microsphere comprises aN,N-methylene-bis-acrylamide monomer (e.g., an ultra-pure monomer) in anamount selected from the group consisting of about 1% by weight, about5% by weight, about 10% by weight, about 15% by weight, about 20% byweight, about 25% by weight, about 30% by weight, about 35% by weight,about 40% by weight, about 45% by weight, about 50% by weight, about 55%by weight, about 60% by weight, about 65% by weight, about 70% byweight, about 75% by weight, about 80% by weight, about 85% by weight,about 90% by weight, and about 95% by weight (or in range thereof).

In one embodiment, the microsphere comprises from about 58% to about 66%by weight of a N-tris-hydroxymethyl methylacrylamide monomer, from about22% to about 26% by weight of a diethylaminoethylacrylamide monomer andabout 6% to about 7% by weight of a N,N-methylene-bis-acrylamidemonomer, and about 0% to about 13% by weight of gelatin or gelatinsubstitute, such that the total is 100%.

The microspheres provided herein can, in certain embodiments, comprisein any combination of the percentages by weight of the above-listedmonomers (e.g., one or more ultra-pure monomers).

In certain embodiments, microspheres provided herein prepared from oneor more of the N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and/or N,N-methylene-bis-acrylamideultra-pure monomers provided herein or comprising one or more of theN-tris-hydroxymethyl methylacrylamide, diethylaminoethylacrylamideand/or N,N-methylene-bis-acrylamide ultra-pure monomer units providedherein (e.g., using a method provided herein) can result in thereduction or one of more side effects in a subject following injectionas compared to the same microsphere prepared without the one or moreultra-pure monomers. Without wishing to be bound by theory, the reducedside effects can be the result of the decreased amount of impurities(e.g., a reaction side product, such as ethanol or acrylic acid,unreacted monomers and/or inhibitors) in the prepared microspheres.

In other embodiments, microspheres provided herein prepared from one ormore of the N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and/or N,N-methylene-bis-acrylamideultra-pure monomers provided herein or comprising one or more of theN-tris-hydroxymethyl methylacrylamide, diethylaminoethylacrylamideand/or N,N-methylene-bis-acrylamide ultra-pure monomer units providedherein (e.g., using a method provided herein) can result in an increasein polymerization efficiency (e.g., wherein more of the monomer isincorporated into the polymer and/or better crosslinking efficiency) ascompared to the same microsphere prepared without the one or moreultra-pure monomers.

In another embodiment, microspheres provided herein prepared from one ormore of the N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and/or N,N-methylene-bis-acrylamideultra-pure monomers provided herein or comprising one or more of theN-tris-hydroxymethyl methylacrylamide, diethylaminoethylacrylamideand/or N,N-methylene-bis-acrylamide ultra-pure monomer units providedherein (e.g., using a method provided herein) can result in an increasein gel point as compared to the same microsphere prepared without theone or more ultra-pure monomers. Increased gel points can result in afaster polymerization reaction, which can further lead to a decrease inthe number of aggregated microspheres in a population. Methods forassessing gel point in polymerization reactions are known in the art(see, e.g., Nita et al. (2007) Rheol. Acta 46, 595-600).

In yet other embodiments, microspheres provided herein prepared from oneor more of the N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and/or N,N-methylene-bis-acrylamideultra-pure monomers provided herein or comprising one or more of theN-tris-hydroxymethyl methylacrylamide, diethylaminoethylacrylamideand/or N,N-methylene-bis-acrylamide ultra-pure monomer units providedherein (e.g., using a method provided herein) can result in animprovement in the overall manufacturing process of the microspheres,which can lead to an improvement in the yield and/or overall quality ofthe microspheres (e.g., less broken and/or aggregated microspheres), amore consistent product, better uniformity in size, narrower sizedistribution, better uniformity in shape, or any combination thereof, ascompared to the same microsphere prepared without the one or moreultra-pure monomers.

4. NMR Analysis of Microsphere

In certain embodiments, the microsphere provided herein, when analyzedby NMR spectroscopy (see e.g., Example 14), exhibits a first peak (e.g.,from about 3.5 ppm to about 4.0 ppm), a second peak (e.g., from about3.0 ppm to about 3.5 ppm, and a third peak (e.g., from about 1.0 ppm toabout 1.4 ppm) in a one-dimensional (1D) ¹H NMR spectrum. In someembodiments, the NMR can be performed by an NMR spectrometer, forexample, by an Avance I Bruker spectrometer (¹H), equipped with a tuned4 mm HR-MAS probehead (¹H, ¹³C, lock ²H) or a similar equipment. Incertain embodiments, the high resolution magic angle spinning (HR-MAS)technique can be employed to analyze the microsphere. Without wishing tobe bound by any theory, the HR-MAS technique was initially developed forthe combinatorial chemistry using the solid phase synthesis. The HR-MASis particularly useful for the analysis of samples not fully soluble orcontaining solids. It gives very good results when the sample has theproperty of swelling or increasing the mobility in an appropriatedsolvent. HR-MAS NMR can be considered a hybrid technique between solidstate NMR and classical solution state NMR. Similar to solid state NMR,the use of magic angle spinning (MAS) effectively removes spectral linebroadening resulting from chemical shift anisotropy, homonuclear dipolarinteractions, and magnetic susceptibility.

In some embodiments, the ¹H NMR spectrum can be recorded at from 100 MHzand 900 MHz, such as at 100 MHz, 200 MHz, 300 MHz, 400 MHz, 500 MHz, 600MHz, 700 MHz, 800 MHz, or 900 MHz, or a range thereof. In a specificembodiment, the ¹H NMR spectrum is recorded at 400 MHz. In certainembodiments, the microsphere can be dispersed in any solvent compatiblewith the NMR analysis. Exemplary solvents include, but are not limitedto, deuterated solvents such as deuterated water, acetic acid-d₄,acetone-d₆, acetonitrile-d₃, benzene-d₆, chloroform-d,dichloromethane-d₂, N,N-dimethyl formamide-d₇, dimethyl sulfoxide-d₆,ethanol-d₆, methanol-d₄, nitromethane-d₃, pyridine-d₅,tetrahydrofuran-d₈, toluene-d₈, trifluoroacetic acid-d₄, andtrifluoroethanol-d₃, and other solvents such as carbon disulphide,1,1,2,2-tetrachloroethane, carbon tetrachloride, diethyl ether (−100°C.), dimethyl ether (−100° C.), 1,4-dioxan, trichlorofluoromethane,nitrobenzene, tetrahydrofuran (−100° C.). In a specific embodiment, thesolvent is deuterated water (D₂O).

In certain embodiments, the microsphere can be analyzed by NMRspectroscopy at a temperature of from about 0° C. to about 80° C., suchas from about 10° C. to about 50° C., from about 15° C. to about 35° C.,from about 20° C. to about 25° C. (e.g., the room temperature), or fromabout 0° C. to about 5° C., or any range thereof. In a specificembodiment, the microsphere is analyzed at a temperature of about 25° C.

In a specific embodiment, the microspheres are analyzed in an aone-dimensional 1D ¹H NMR spectrum, using the parameters essentially asprovided in Example 14 (e.g., Table 10), and the first, second and thirdpeaks and/or the integration ratios of the second peak to the first peak(normalized to 1) and the third peak to the first peak are determined.

The first peak exhibited by the microsphere in the ¹H NMR spectrum maybe attributed, for example, to the tris-hydroxymethyl groups (e.g.,C(CH₂OH)₃) in the copolymer of the microsphere. In certain embodiments,the first peak exhibited by the microsphere in the ¹H NMR spectrum isfrom about 3.60 ppm to about 3.95 ppm, such as from about 3.65 ppm toabout 3.90 ppm, from about 3.70 ppm to about 3.85 ppm, or from about3.75 ppm to about 3.80 ppm, or any range thereof. In a specificembodiment, the first peak exhibited by the microsphere in the ¹H NMRspectrum is at about 3.77 ppm.

The second peak exhibited by the microsphere in the ¹H NMR spectrum maybe attributed, for example, to the CH₂ groups linked to the basicnitrogen atom (e.g., (CH₂N(CH₂CH₃)₂)). In certain embodiments, thesecond peak exhibited by the microsphere in the ¹H NMR spectrum is fromabout 3.05 ppm to about 3.45 ppm, such as from about 3.10 ppm to about3.40 ppm, from about 3.15 ppm to about 3.35 ppm, from about 3.20 ppm toabout 3.30 ppm, or from about 3.15 ppm to about 3.25 ppm, or any rangethereof. In a specific embodiment, the second peak exhibited by themicrosphere in the ¹H NMR spectrum is at about 3.2 ppm.

The third peak exhibited by the microsphere in the ¹H NMR spectrum maybe attributed, for example, to the groups in the β position of thecarboxamide group in the polymerized structure of the microsphere (e.g.,CH₂—CHCONH). In certain embodiments, the third peak exhibited by themicrosphere in the ¹H NMR spectrum is from about 1.01 ppm to about 1.49ppm, such as from about 1.05 ppm to about 1.47 ppm, from about 1.10 ppmto about 1.45 ppm, from about 1.15 ppm to about 1.40 ppm, or from about1.25 ppm to about 1.35 ppm or any range thereof. In a specificembodiment, the third peak exhibited by the microsphere in the ¹H NMRspectrum is at about 1.3 ppm. In some embodiments, (i) the second peakis at about 3.245 ppm or less, such as about 3.211 ppm or less or about3.192 ppm or less; (ii) the third peak is at about 1.297 ppm or less,such as about 1.283 ppm or less or about 1.276 ppm or less; (ii) or anycombination of (i) and (ii). In one embodiment, the second peak is notat 3.212 or 3.246 (+/−0.002 or 0.005) ppm and/or the third peak is notat 1.284 or 1.298 (+/−0.002 or 0.005) ppm.

In certain embodiments, the integration ratio of the second peak to thefirst peak exhibited by the microsphere in the ¹H NMR spectrum is fromabout 0.498 to about 0.650, such as from about 0.53 to about 0.63, fromabout 0.55 to about 0.60, or any range thereof. In some embodiments, theintegration ratio of the second peak to the first peak exhibited by themicrosphere in the ¹H NMR spectrum is about 0.50, 0.51, 0.52, 0.53,0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or0.65, or any range thereof. In certain embodiments, the integrationratio of the second peak to the first peak exhibited by the microspherein the ¹H NMR spectrum is about 0.571, 0.572, 0.573, 0.574, 0.575,0.576, 0.577, 0.578, or 0.579, or any range thereof. In a specificembodiment, the integration ratio of the second peak to the first peakexhibited by the microsphere in the ¹H NMR spectrum is about 0.574.

Integration ratios can be calculated using methods known in the art, forexample, by normalizing the first peak to the value of 1 and themcomparing the ratio of the second (or third) peak to the first peak.Without wishing to be bound by theory, it is thought that theintegration ratios can be used to directly correlate to the proportionof each material that is incorporated into the polymer chain. That is,higher integration ratios can correlate with more monomers and/or ahigher proportion of monomers and less impurities incorporated into thefinal polymer product, as well as an overall better efficiency ofpolymerization.

In some embodiments, the integration ratio of the second peak to thefirst peak exhibited by a microsphere comprising a copolymer prepared bycopolymerizing (i) an ultra-pure N-tris-hydroxymethyl methylacrylamidemonomer, (ii) an ultra pure diethylaminoethylacrylamide monomer, (iii)an ultra-pure N,N-methylene-bis-acrylamide monomer, (iv) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer and an ultra purediethylaminoethylacrylamide monomer, (v) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer and an ultra-pure anN,N-methylene-bis-acrylamide monomer, (vi) an ultra purediethylaminoethylacrylamide monomer and an ultra-pure anN,N-methylene-bis-acrylamide monomer, or (vii) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer, an ultra purediethylaminoethylacrylamide monomer, and an ultra-pure anN,N-methylene-bis-acrylamide monomer in the ¹H NMR spectrum is fromabout 5% to about 100% higher, such as about 5% higher, about 10%higher, about 15% higher, about 20% higher, about 25% higher, about 30%higher, about 35% higher, about 40% higher, about 45% higher, about 50%higher, about 55% higher, about 60% higher, about 65% higher, about 70%higher, about 75% higher, about 80% higher, about 85% higher, about 90%higher, about 95% higher, about 100% higher, or any range thereof, ascompared to the same microsphere prepared without the ultra-puremonomer(s).

In some embodiments, the integration ratio of the second peak to thefirst peak exhibited by a microsphere comprising (i) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit, (ii) an ultra purediethylaminoethylacrylamide monomer unit, (iii) an ultra-pureN,N-methylene-bis-acrylamide monomer unit, (iv) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit and an ultra purediethylaminoethylacrylamide monomer unit, (v) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit, (vi) an ultra purediethylaminoethylacrylamide monomer unit and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit, or (vii) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit, an ultra purediethylaminoethylacrylamide monomer unit, and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit in the ¹H NMR spectrum is fromabout 5% to about 100% higher, such as about 5% higher, about 10%higher, about 15% higher, about 20% higher, about 25% higher, about 30%higher, about 35% higher, about 40% higher, about 45% higher, about 50%higher, about 55% higher, about 60% higher, about 65% higher, about 70%higher, about 75% higher, about 80% higher, about 85% higher, about 90%higher, about 95% higher, about 100% higher, or any range thereof, ascompared to the same microsphere that do not comprise the ultra-puremonomer unit(s) (prepared without the respective ultra-pure monomer(s)).

In certain embodiments, the integration ratio of the third peak to thefirst peak exhibited by the microsphere in the ¹H NMR spectrum is fromabout 0.53 to about 0.75, such as from about 0.57 to about 0.60, fromabout 0.60 to about 0.65, from about 0.61 to about 0.75 or from about0.61 to about 0.65 or any range thereof. In certain embodiments, theintegration ratio of the third peak to the first peak exhibited by themicrosphere in the ¹H NMR spectrum is about 0.53, 0.54, 0.55, 0.56,0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68,0.69, 0.70, 0.71, 0.72, 0.73, 0.74, or 0.75, or any range thereof. Incertain embodiments, the integration ratio of the third peak to thefirst peak exhibited by the microsphere in the ¹H NMR spectrum is about0.611, 0.612, 0.613, 0.614, 0.615, 0.616, 0.617, 0.618, 0.619, 0.620,0.621, 0.622, 0.623, 0.624, 0.625, 0.626, 0.627, 0.628, or 0.629, or anyrange thereof. In a specific embodiment, the integration ratio of thethird peak to the first peak exhibited by the microsphere in the ¹H NMRspectrum is about 0.625.

In some embodiments, the integration ratio of the third peak to thefirst peak exhibited by a microsphere comprising a copolymer prepared bycopolymerizing (i) an ultra-pure N-tris-hydroxymethyl methylacrylamidemonomer, (ii) an ultra pure diethylaminoethylacrylamide monomer, (iii)an ultra-pure an N,N-methylene-bis-acrylamide monomer, (iv) anultra-pure N-tris-hydroxymethyl methylacrylamide monomer and an ultrapure diethylaminoethylacrylamide monomer, (v) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer and an ultra-pure anN,N-methylene-bis-acrylamide monomer, (vi) an ultra purediethylaminoethylacrylamide monomer and an ultra-pure anN,N-methylene-bis-acrylamide monomer or (vii) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer, an ultra purediethylaminoethylacrylamide monomer, and an ultra-pure anN,N-methylene-bis-acrylamide monomer in the ¹H NMR spectrum is fromabout 5% to about 100% higher, such as about 5% higher, about 10%higher, about 15% higher, about 20% higher, about 25% higher, about 30%higher, about 35% higher, about 40% higher, about 45% higher, about 50%higher, about 55% higher, about 60% higher, about 65% higher, about 70%higher, about 75% higher, about 80% higher, about 85% higher, about 90%higher, about 95% higher, about 100% higher, or any range thereof, ascompared to the same microsphere prepared without the ultra-puremonomer(s).

In some embodiments, the integration ratio of the third peak to thefirst peak exhibited by a microsphere comprising (i) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit, (ii) an ultra purediethylaminoethylacrylamide monomer unit, (iii) an ultra-pureN,N-methylene-bis-acrylamide monomer unit, (iv) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit and an ultra purediethylaminoethylacrylamide monomer unit, (v) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit, (vi) an ultra purediethylaminoethylacrylamide monomer unit and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit, or (vii) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit, an ultra purediethylaminoethylacrylamide monomer unit, and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit in the ¹H NMR spectrum is fromabout 5% to about 100% higher, such as about 5% higher, about 10%higher, about 15% higher, about 20% higher, about 25% higher, about 30%higher, about 35% higher, about 40% higher, about 45% higher, about 50%higher, about 55% higher, about 60% higher, about 65% higher, about 70%higher, about 75% higher, about 80% higher, about 85% higher, about 90%higher, about 95% higher, about 100% higher, or any range thereof, ascompared to the same microsphere that do not comprise the ultra-puremonomer unit(s) (prepared without the respective ultra-pure monomer(s)).

In some embodiments, (a) the integration ratio of the second peak to thefirst peak exhibited by a microsphere comprising a copolymer prepared bycopolymerizing (i) an ultra-pure N-tris-hydroxymethyl methylacrylamidemonomer, (ii) an ultra pure diethylaminoethylacrylamide monomer, (iii)an ultra-pure an N,N-methylene-bis-acrylamide monomer, (iv) anultra-pure N-tris-hydroxymethyl methylacrylamide monomer and an ultrapure diethylaminoethylacrylamide monomer, (v) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer and an ultra-pure anN,N-methylene-bis-acrylamide monomer, (vi) an ultra purediethylaminoethylacrylamide monomer and an ultra-pure anN,N-methylene-bis-acrylamide monomer or (vii) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer, an ultra purediethylaminoethylacrylamide monomer, and an ultra-pure anN,N-methylene-bis-acrylamide monomer in the ¹H NMR spectrum is fromabout 5% to about 100% higher, such as about 5% higher, about 10%higher, about 15% higher, about 20% higher, about 25% higher, about 30%higher, about 35% higher, about 40% higher, about 45% higher, about 50%higher, about 55% higher, about 60% higher, about 65% higher, about 70%higher, about 75% higher, about 80% higher, about 85% higher, about 90%higher, about 95% higher, about 100% higher, or any range thereof, and(b) the integration ratio of the third peak to the first peak exhibitedby a microsphere comprising a copolymer prepared by copolymerizing (i)an ultra-pure N-tris-hydroxymethyl methylacrylamide monomer, (ii) anultra pure diethylaminoethylacrylamide monomer, (iii) an ultra-pure anN,N-methylene-bis-acrylamide monomer, (iv) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer and an ultra purediethylaminoethylacrylamide monomer, (v) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer and an ultra-pure anN,N-methylene-bis-acrylamide monomer, (vi) an ultra purediethylaminoethylacrylamide monomer and an ultra-pure anN,N-methylene-bis-acrylamide monomer or (vii) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer, an ultra purediethylaminoethylacrylamide monomer, and an ultra-pure anN,N-methylene-bis-acrylamide monomer, respectively, in the ¹H NMRspectrum is from about 5% to about 100% higher, such as about 5% higher,about 10% higher, about 15% higher, about 20% higher, about 25% higher,about 30% higher, about 35% higher, about 40% higher, about 45% higher,about 50% higher, about 55% higher, about 60% higher, about 65% higher,about 70% higher, about 75% higher, about 80% higher, about 85% higher,about 90% higher, about 95% higher, about 100% higher, or any rangethereof; and (b)

In other embodiments, (a) the integration ratio of the second peak tothe first peak exhibited by a microsphere comprising (i) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit, (ii) an ultra purediethylaminoethylacrylamide monomer unit, (iii) an ultra-pureN,N-methylene-bis-acrylamide monomer unit, (iv) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit and an ultra purediethylaminoethylacrylamide monomer unit, (v) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit, (vi) an ultra purediethylaminoethylacrylamide monomer unit and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit, or (vii) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit, an ultra purediethylaminoethylacrylamide monomer unit, and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit in the ¹H NMR spectrum is fromabout 5% to about 100% higher, such as about 5% higher, about 10%higher, about 15% higher, about 20% higher, about 25% higher, about 30%higher, about 35% higher, about 40% higher, about 45% higher, about 50%higher, about 55% higher, about 60% higher, about 65% higher, about 70%higher, about 75% higher, about 80% higher, about 85% higher, about 90%higher, about 95% higher, about 100% higher, or any range thereof, and(b) the integration ratio of the third peak to the first peak exhibitedby a microsphere comprising (i) an ultra-pure N-tris-hydroxymethylmethylacrylamide monomer unit, (ii) an ultra purediethylaminoethylacrylamide monomer unit, (iii) an ultra-pureN,N-methylene-bis-acrylamide monomer unit, (iv) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit and an ultra purediethylaminoethylacrylamide monomer unit, (v) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit, (vi) an ultra purediethylaminoethylacrylamide monomer unit and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit, or (vii) an ultra-pureN-tris-hydroxymethyl methylacrylamide monomer unit, an ultra purediethylaminoethylacrylamide monomer unit, and an ultra-pure anN,N-methylene-bis-acrylamide monomer unit in the ¹H NMR spectrum is fromabout 5% to about 100% higher, such as about 5% higher, about 10%higher, about 15% higher, about 20% higher, about 25% higher, about 30%higher, about 35% higher, about 40% higher, about 45% higher, about 50%higher, about 55% higher, about 60% higher, about 65% higher, about 70%higher, about 75% higher, about 80% higher, about 85% higher, about 90%higher, about 95% higher, about 100% higher, or any range thereof, ascompared to the same microsphere that do not comprise the ultra-puremonomer unit(s) (prepared without the respective ultra-pure monomer(s)).

Also provided herein is a microsphere comprising (a) a copolymerprepared by copolymerizing a N-tris-hydroxymethyl methylacrylamidemonomer, a diethylaminoethylacrylamide monomer and aN,N-methylene-bis-acrylamide monomer, wherein one, two or three of themonomers is an ultra-pure monomer, and (b) crosslinked gelatin; whereinthe microsphere exhibits in a ¹H NMR spectrum, a first peak from about3.5 ppm to about 4 ppm, a second peak from about 3 ppm to about 3.5 ppm,and a third peak from about 1 ppm to about 1.5 ppm; and wherein (i) theintegration ratio of the second peak to the first peak is higher thanthe integration ratio of the same microsphere prepared without theultra-pure monomer(s), (ii) the integration ratio of the third peak tothe first peak is higher than the integration ratio of the samemicrosphere prepared without the ultra-pure monomer(s), or (iii) theintegration ratio of the second peak to the first peak and theintegration ratio of the third peak to the first peak are each higherthan the respective integration ratios of same microsphere preparedwithout the ultra-pure monomer(s). In some embodiments, the integrationratio of the second peak to the first peak and/or the integration ratioof the third peak to the first peak is independently selected from about5% higher, about 10% higher, about 15% higher, about 20% higher, about25% higher, about 30% higher, about 35% higher, about 40% higher, about45% higher, about 50% higher, about 55% higher, about 60% higher, about65% higher, about 70% higher, about 75% higher, about 80% higher, about85% higher, about 90% higher, about 95% higher, about 100% higher, orany range thereof.

Also provided herein is a microsphere comprising (a) a copolymercomprising a N-tris-hydroxymethyl methylacrylamide monomer unit, adiethylaminoethylacrylamide monomer unit and aN,N-methylene-bis-acrylamide monomer unit, wherein one, two or three ofthe monomers units is an ultra-pure monomer unit, and (b) crosslinkedgelatin; wherein the microsphere exhibits in a ¹H NMR spectrum, a firstpeak from about 3.5 ppm to about 4 ppm, a second peak from about 3 ppmto about 3.5 ppm, and a third peak from about 1 ppm to about 1.5 ppm;and wherein (i) the integration ratio of the second peak to the firstpeak is higher than the integration ratio of the same microsphere thatdoes not comprise the ultra-pure monomer unit(s) (prepared without therespective ultra-pure monomer(s)), (ii) the integration ratio of thethird peak to the first peak is higher than the integration ratio of thesame microsphere p same microsphere that does not comprise theultra-pure monomer unit(s) (prepared without the respective ultra-puremonomer(s)), or (iii) the integration ratio of the second peak to thefirst peak and the integration ratio of the third peak to the first peakare each higher than the respective integration ratios of samemicrosphere that does not comprise the ultra-pure monomer unit(s)(prepared without the respective ultra-pure monomer(s)). In someembodiments, the integration ratio of the second peak to the first peakand/or the integration ratio of the third peak to the first peak isindependently selected from about 5% higher, about 10% higher, about 15%higher, about 20% higher, about 25% higher, about 30% higher, about 35%higher, about 40% higher, about 45% higher, about 50% higher, about 55%higher, about 60% higher, about 65% higher, about 70% higher, about 75%higher, about 80% higher, about 85% higher, about 90% higher, about 95%higher, about 100% higher, or any range thereof.

In specific embodiments, the microsphere is an ultra-pure microsphere.In certain embodiments, the ultra-pure microsphere comprises 10% orless, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% orless, 3% or less, 2% or less, 1% or less or 0% of impurities by weight.In some embodiments, the ultra-pure microsphere comprise 90% or more,91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more, 99% or more, or 100% by weight ofN-tris-hydroxymethyl methylacrylamide, diethylaminoethylacrylamide,N,N-methylene-bis-acrylamide and gelatin.

In one embodiment, provided herein is a microsphere comprising: (a) acopolymer comprising a N-tris-hydroxymethyl methylacrylamide monomerunit, a diethylaminoethylacrylamide monomer unit and aN,N-methylene-bis-acrylamide monomer unit, and (b) crosslinked gelatin;wherein the microsphere exhibits in a ¹H NMR spectrum, a first peak fromabout 3.5 ppm to about 4 ppm, a second peak from about 3 ppm to about3.5 ppm, and a third peak from about 1 ppm to about 1.5 ppm; and wherein(i) the integration ratio of the second peak to the first peak is from0.50 to about 0.65, (ii) the integration ratio of the third peak to thefirst peak is from 0.61 to about 0.75, or (iii) a combination of (i) and(ii). In one embodiments, the first peak is at about 3.77 ppm, thesecond peak is at about 3.2 ppm, the third peak is at about 1.3 ppm, ora combination thereof. In some embodiments, the integration ratio of thesecond peak to the first peak is about 0.574, or wherein the integrationratio of the third peak to the first peak is about 0.625. In oneembodiments, the microsphere of claim 1, wherein the microsphere is at25° C. and/or in a deuterated solvent when the ¹H NMR spectrum isrecorded and/or the ¹H NMR spectrum is recorded at 400 MHz. In someembodiments, one, two or three of the N-tris-hydroxymethylmethylacrylamide, diethylaminoethylacrylamide andN,N-methylene-bis-acrylamide monomer units is an ultra-pure monomerunit. In certain embodiments, the N-tris-hydroxymethyl methylacrylamidemonomer unit comprises less than 9% of impurities and/or thediethylaminoethylacrylamide monomer unit comprises less than 2% ofimpurities (e.g., as determined by the bromine test). In certainembodiments, the N-tris-hydroxymethyl methylacrylamide monomer unitcomprises less than 9% of impurities and/or thediethylaminoethylacrylamide monomer unit comprises less than 2% ofimpurities (e.g., as determined by HPLC). In another embodiment, themicrosphere has a diameter from about 1 μm to about 2000 from about 40μm to about 120 from about 100 μm to about 300 from about 300 μm toabout 500 from about 500 μm to about 700 from about 700 μm to about 900or from about 900 μm to about 1200 μm.

B. Compositions

The microspheres provided herein can be used in a composition (e.g.,pharmaceutical composition) with a pharmaceutically acceptable liquid orother biocompatible carrier.

In certain embodiments, the microspheres or the microspheres in thecompositions are substantially uniform in size. For example, in certainembodiments, the difference in diameter between individual microspheresis from about 0 μm to about 100 from about 0 μm to about or from about 0μm to about 25 In some embodiments, the microspheres have differences indiameter of 100 μm or less, about 50 μm or less, about 25 mm or less,about 10 mm or less or about 5 μm or less.

In certain embodiments, the microspheres in the compositions have adiameter from about 1 μm to 2000 from about 10 μm to 1000 from about 40μm to about 120 from about 100 μm to about 300 from about 300 μm toabout 500 from about 500 μm to about 700 from about 700 μm to about 900or from about 900 μm to about 1200 μm.

In certain embodiments, the microspheres are in a population whereingreater than 68% have a diameter of ±20% of the mean, ±10% of the mean,or ±5% of the mean diameter. In one embodiment, the microspheres are ina population wherein greater than 75% have a diameter of ±20% of themean, ±15% of the mean, ±10% of the mean or ±5% of the mean diameter, ora range thereof.

The composition can be in the form of a suspension, a hydrogel, or anemulsion. The composition can also be a suspension of microspheres inthe liquid. The hydrophilic nature of the microspheres permits placingthem in suspension, and in particular, in the form of sterile andnon-pyrogenic or pyrogen-free injectable solutions, while avoiding theformation of aggregates or adhesion to the walls of storage containersand implantation devices, such as catheters, syringes, needles, and thelike.

In some embodiments, a catheter is used for the administration. In aspecific embodiment, a selectively positioned catheter is used. Suchcatheter has a guiding catheter inserted into a main artery, amicrocatheter with or without a steerable microguide wire, and ahemostatic valve that provides a tight seal between the guiding catheterand the microcatheter, so that a continuous heparin flush can be used toprevent blood clotting.

In specific embodiments, the microspheres or the pharmaceuticalcompositions are suitable for injection. In specific embodiments, themicrospheres and/or compositions comprising the microspheres aresterile. The microspheres can be sterilized by any method known in theart, for example, by irradiation, such as gamma or beta irradiation. Incertain embodiments, the microspheres are prepared aseptically usingaseptic techniques. In some embodiments, the microspheres preparedaseptically comprise a therapeutic agent or drug.

The pharmaceutically acceptable liquid can be, without limitation,saline, a buffer-solution, water, an isotonic solution, a biologicalfluid or a mixture thereof. The liquid can also be a salt solution, and,in certain embodiments, is composed of cations selected from the groupconsisting of sodium, potassium, calcium, magnesium, iron, zinc, andammonium, for example, in an amount of from about 0.01 M to about 5 M.In certain embodiments, the microspheres are suspended in, or otherwiseadministered to a subject in combination with lipiodol and optionally adrug (e.g., doxorubicin) or other therapeutic agent.

The composition can comprise the microspheres in an amount from about10% to about 90% by weight and the liquid (or other biocompatiblecarrier) in an amount from about 10% to about 90% by weight. Thecomposition can also comprise the microspheres in an amount from about10% to about 50% by weight and the liquid (or other biocompatiblecarrier) in an amount from about 50% to about 90% by weight.

Acceptable pharmaceutical carriers for therapeutic use include diluents,solubilizers, lubricants, suspending agents, encapsulating materials,solvents, thickeners, dispersants, buffers such as phosphate, citrate,acetate and other organic acid salts, anti-oxidants such as ascorbicacid, preservatives, low molecular weight (less than about 10 residues)peptides such as polyarginine, proteins such as serum albumin, gelatinor immunoglobulins, hydrophilic polymers such aspoly(vinylpyrrolindinone), amino acids such as glycine, glutamic acid,aspartic acid or arginine, monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannoseor dextrines, chelating agents such as EDTA, sugar alcohols such asmannitol or sorbitol, counter-ions such as sodium and/or non-ionicsurfactants such as tween, pluronics or PEG.

In some embodiments, the composition or formulation comprises themicrospheres provided herein, for example as a dry powder, which can beprovided in a container, such as a vial or syringe. The microspheres canbe mixed with one or more pharmaceutical carriers and/or one or moreother components provided herein (e.g., a marking agent and/or atherapeutic agent, such as a drug) provided herein prior to, during orafter use (e.g., before, during or after injection into a patient).

In some embodiments, the biocompatible carrier is an aqueous-basedsolution, a hydro-organic solution, an organic solution, a non-aqueoussolution, or a mixture thereof. In certain embodiments, thebiocompatible carrier comprises a salt composed of cations, such assodium, potassium, calcium, magnesium, iron, zinc, ammonium, andmixtures thereof, for example, in an amount of from about 0.01 M toabout 5 M.

In certain embodiments, the microspheres of the composition are visiblein the light and within the body, for example, by further comprising amarking agent. In some embodiment, microspheres can be marked aftertheir synthesis. This can be done, for example, by grafting offluorescent markers derivatives (including, for example, fluoresceinisothiocyanate (FITC), rhodamine isothiocyanate (RITC) and the like). Insome embodiments, a detectable monomer can be obtained by chemicalcoupling of the monomer with a marker, which can be: a chemical dye,such as Cibacron Blue or Procion Red HE-3B, making possible a directvisualization of the microspheres, a magnetic resonance imaging agent(erbium, gadolinium or magnetite); a contrasting agent, such as bariumor iodine salts, (including, for example,(acrylamido-3-propionamido)-3-triiodo-2,4,6-benzoic acid. In the case ofbarium or magnetite salts, they can be directly introduced in poweredform in the initial monomer solution.

In a certain embodiment, the composition comprises a contrast agent orother marking agent, such as a radiopaque contrast agent, such as anon-ionic contrast agent. In some embodiments, the contrast agent ismixed with the microspheres in the composition prior to, during and/orafter injection into the patient. In a specific embodiment, the patientis administered a composition comprising a contrast agent (e.g., anon-ionic contrast agent) and microspheres provided herein.

C. Method of Making Microspheres

Provided herein a method for producing microspheres comprising a gelatinor gelatin substitute and a copolymer of a N-tris-hydroxymethylmethylacrylamide, a diethylaminoethylacrylamide and aN,N-methylene-bis-acrylamide. Further provided herein, for example, aremicrospheres produced by this method, as well as compositions and usesof the microspheres thereof.

The microspheres provided herein can be prepared by any method known toa skilled artisan, for example, by suspension polymerization (e.g., awater-in-oil suspension or emulsion), drop-by-drop polymerization, asdescribed in E. Boschetti, Microspheres for Biochromatography andBiomedical Applications. Part I, Preparation of Microbeads In:Microspheres, Microencapsulation and Liposomes, John Wiley & Sons,Arshady R., Ed., vol. 2, p. 171-189 (1999), or as in U.S. Pat. Nos.5,635,215 and 5,648,100, each of which is incorporated herein byreference. The mode of microsphere preparation selected will usuallydepend upon the desired characteristics, such as microsphere diameterand chemical composition, for the resulting microspheres. In someembodiments of the various methods provided herein, the monomers arepolymerized by radical polymerization or radiation.

In certain embodiments, polymerization is carried out by emulsion orsuspension polymerization, since it makes it possible to access directlymicrospheres of a desired size. It can be conducted as follows: theaqueous solution containing the different dissolved constituents (e.g.,different monomers, cell adhesion agent), is mixed by stirring, with asolution comprising a liquid organic phase (e.g., vegetable, animal ormineral oils, certain petroleum distillation products, chlorinatedhydrocarbons or a mixture of these different solutions) that has low(e.g., about 15% or less, about 10% or less, about 5% or less, about 4%or less, about 3% or less, about 2% or less, about 1% or less, about0.5%, about 0.1% or less, or about 0%) miscibility in water (e.g., isimmiscible) at 25° C., and optionally in the presence of an emulsifier(e.g., a sorbitan sesquioleate) to create a suspension of droplets,which are then turned into solid gel by polymerization of monomers bymeans of appropriate catalysts. In a specific embodiment, the liquidorganic phase has a miscibility of about 1% or less in water at 25° C.In a certain embodiments, the liquid organic phase comprises or consistsof an oil. In a specific embodiment, the liquid organic phase comprisesor consists of a vegetable oil or a mineral oil (e.g., paraffin oil) ora combination of a vegetable oil and a mineral oil. The rate of stirringis adjusted so as to obtain an aqueous phase emulsion in the organicphase forming drops of desired diameter. Polymerization is then startedoff by addition of the initiator. It is accompanied by an exothermicreaction and its development can then be followed by measuring thetemperature of the reaction medium.

The polymerization initiator is advantageously chosen among the redoxsystems, for example, using combinations of an alkali metal persulfatewith N,N,N′,N′-tetramethylethylenediamine or withdimethylaminopropionitrile, organic peroxides such as benzoyl peroxidesor even 2,2′-azo-bis-isobutyronitrile. The quantity of initiator used isadapted to the quantity of monomers and the rate of polymerizationsought. Polymerization can be carried out in mass or in emulsion. In thecase of a mass polymerization, the aqueous solution containing thedifferent dissolved constituents and the initiator undergoespolymerization in a homogeneous medium. This makes it possible to accessa lump of aqueous gel which can then be separated into microspheres, bypassing, for example, through the mesh of a screen. In certainembodiments, a polymerization initiator is added in the aqueous phasebefore emulsification when the polymerization initiator includes severalcomponents (redox system).

The microspheres thus obtained can then be recovered by cooling,decanting and filtration. They are then separated by size category andwashed to eliminate any trace of secondary product.

The polymerization stage can be followed by a stage of reticulation ofthe cell adhesion agent and possibly by a marking agent stage in thecase of microspheres rendered identifiable by grafting after synthesis.For example, in certain embodiments, radical polymerization of theN-tris-hydroxymethyl methylacrylamide, diethylaminoethylacrylamide, andN,N-methylene-bis-acrylamide monomers is followed by heating at a highertemperature, such as 37° C., such that the gelatin in the microspheresbecomes water soluble. Then, a crosslinker, such as glutaraldehyde isused to crosslink the gelatin (e.g., about 300 ml of glutaraldehyde perabout 1 L of microspheres). In certain embodiments, the microspheres aresubjected to sonication prior to gelatin crosslinking.

Thus, in one aspect, provided herein is a method of making amicrosphere, comprising: (a) preparing an aqueous solution comprising aN-tris-hydroxymethyl methylacrylamide monomer, adiethylaminoethylacrylamide monomer, a N,N-methylene-bis-acrylamidemonomer, and gelatin, wherein at least one of the monomers is anultra-pure monomer; (b) adding the aqueous solution to a liquid organicphase (e.g., an oil, such as a mineral oil, such as a paraffin oil) thathas low miscibility in water, before or while stirring; therebyproducing microspheres comprising a copolymer comprisingN-tris-hydroxymethyl methylacrylamide, diethylaminoethylacrylamide andN,N-methylene-bis-acrylamide monomer units; (c) optionally subjectingthe microspheres to sonication (e.g., ultrasonication); and (d)crosslinking the gelatin. Subsequently, microspheres can be collected byfiltration or centrifugation and washed. In certain embodiments, theaqueous solution of monomers can contains adhesion agents such ascollagen (gelatin is a denatured collagen).

In another aspect, provided herein are microspheres prepared by aprocess comprising: (a) preparing an aqueous solution comprising (i) aN-tris-hydroxymethyl methylacrylamide monomer, (ii) adiethylaminoethylacrylamide monomer, (iii) aN,N-methylene-bis-acrylamide monomer, and (iv) gelatin, wherein theN-tris-hydroxymethyl methylacrylamide monomer, thediethylaminoethylacrylamide monomer and/or theN,N-methylene-bis-acrylamide monomer is an ultra-pure monomer; (b) aadding the aqueous solution to a liquid organic phase (e.g., an oil,such as a mineral oil, such as a paraffin oil) that has low miscibilityin water, before or while stirring; thereby producing microspherescomprising a copolymer comprising N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and N,N-methylene-bis-acrylamide monomerunits; (c) optionally subjecting the microspheres to sonication (e.g.,ultrasonication); and (d) crosslinking the gelatin. In some embodiments,the microsphere exhibits in a ¹H NMR spectrum (e.g., as provided inExample 14), a first peak (e.g., from about 3.5 ppm to about 4 ppm), asecond peak (e.g., from about 3 ppm to about 3.5 ppm), and a third peak(e.g., from about 1 ppm to about 1.5 ppm). In specific embodiments, theintegration ratio of the second peak to the first peak is about 0.50 toabout 0.65 and/or the integration ratio of the third peak to the firstpeak is about 0.55 to about 0.75 (e.g., about 0.61 to about 0.75). Incertain embodiments, the microspheres are uniform in size. In otherembodiments, the microspheres have a diameter from about 1 μm to 2000μm, from 40 μm to about 120 μm, from about 100 μm to about 300 μm, fromabout 300 μm to about 500 μm, from about 500 μm to about 700 μm, fromabout 700 μm to about 900 μm, or from about 900 μm to about 1200 μm. Inspecific embodiments, less than 1% of the microspheres are aggregated(sticking) microspheres.

Also provided herein is a method of making ultra-pure microspherecomprising about 10% or less, 9% or less, 8% or less, 7% or less, 6% orless, 5% or less, 4% or less, 3% or less, 2% or less or 1% or less ofimpurities and/or comprises 90% or more, 91% or more, 92% or more, 93%or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or moreor 99% or more by weight of a N-tris-hydroxymethyl methylacrylamideultra-pure monomer, a diethylaminoethylacrylamide ultra-pure monomer, aN,N-methylene-bis-acrylamide ultra-pure monomer, and gelatin, bysuspension polymerization, for example by (a) preparing an aqueoussolution comprising (i) a N-tris-hydroxymethyl methylacrylamide monomer,(ii) a diethylaminoethylacrylamide monomer, (iii) aN,N-methylene-bis-acrylamide monomer, and (iv) gelatin, one, two orthree of the N-tris-hydroxymethyl methylacrylamide monomer and/or thediethylaminoethylacrylamide monomer or N,N-methylene-bis-acrylamide isan ultra-pure monomer; (b) adding the aqueous solution to a liquidorganic phase (e.g., an oil, such as a mineral oil, such as a paraffinoil) that has low miscibility in water, before or while stirring;thereby producing microspheres comprising a copolymer comprisingN-tris-hydroxymethyl methylacrylamide, diethylaminoethylacrylamide andN,N-methylene-bis-acrylamide monomer units; (c) optionally subjectingthe microspheres to ultrasonication; and (d) crosslinking the gelatin.Also provided herein are the ultra-pure microspheres comprising therespective monomer units prepared by this process.

In certain embodiments of the various methods provided herein, theaqueous solution is added through a feed ring to the liquid organicphase.

In some embodiments of the methods provided herein, the method furthercomprises sieving the microspheres (e.g., for size calibration and/oreto reduce the number of aggregated microspheres in a population ofmicrospheres). In other embodiments, the method does not comprisesubjecting the microspheres to one or more rounds of sieving (e.g., forsize calibration and/ore to reduce the number of aggregated microspheresin a population of microspheres).

In specific embodiments, the methods provided herein comprise subjectingthe microspheres to sonication (e.g., ultrasonication). In certainembodiments, the sonication is done in an ultrasonic bath, e.g., forfrom about 10 minutes to about 15 minutes. In specific embodiments, theN-tris-hydroxymethyl methylacrylamide monomer comprises less than 9% ofimpurities and/or the diethylaminoethylacrylamide monomer comprises lessthan 2% of impurities.

In certain embodiments of the methods provided herein, the use of one ormore of N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and N,N-methylene-bis-acrylamide ultra-puremonomers provided herein results in a higher yield of microsphereproduct as compared to a method that does not use the one or moreultra-pure monomers.

In other embodiments of the methods provided herein, the use of one ormore of N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and N,N-methylene-bis-acrylamide ultra-puremonomers provided herein results in a narrower size distribution ofproduced microspheres as compared to a method that does not use the oneor more ultra-pure monomers.

In other embodiments of the methods provided herein, the use of one ormore of N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and N,N-methylene-bis-acrylamide ultra-puremonomers provided herein results in an increase in polymerizationefficiency (e.g., wherein more of the monomer is incorporated into thepolymer and/or better crosslinking efficiency) as compared to the samemicrosphere prepared without the one or more ultra-pure monomers.

In another embodiment of the methods provided herein, the use of one ormore of N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and N,N-methylene-bis-acrylamide ultra-puremonomers provided herein results in an increase in gel point as comparedto the same microsphere prepared without the one or more ultra-puremonomers. Increased gel points can result in a faster polymerizationreaction, which can further lead to a decrease in the number ofaggregated microspheres in a population. Methods for assessing gel pointin polymerization reactions are known in the art (see, e.g., Nita et al.(2007) Rheol. Acta 46, 595-600).

In yet other embodiments of the methods provided herein, the use of oneor more of N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide and N,N-methylene-bis-acrylamide ultra-puremonomers provided herein results in an improvement in the overallmanufacturing process of the microspheres, which can lead to animprovement in the yield and/or overall quality of the microspheres(e.g., less broken and/or aggregated microspheres), a more consistentproduct, better uniformity in size, narrower size distribution, betteruniformity in shape, or any combination thereof, as compared to the samemicrosphere prepared without the one or more ultra-pure monomers.

1. Feed Ring Process

In certain embodiments, the microspheres provided herein are producedusing Feed Ring equipment (e.g., as shown in FIG. 6) during the step ofadding the aqueous solution to a liquid organic phase (e.g., an oil,such as a mineral oil, such as a paraffin oil) that has low miscibilityin water, before or while stirring (e.g., in step (b) in certain of themethods provided herein). The Feed Ring process can be used to furtherimprove the quality of the microspheres produced, for example, byremoving some side reaction leading to encapsulation of microspheres,encapsulation of oil and/or limiting the number of broken spheres in apopulation.

In an illustrative embodiment, Feed Ring equipment can comprise a staticmixer. In one embodiment, the static mixer comprises a longitudinal axisand a comb having a plurality of spaced fingers extending outward from across-beam connected to the longitudinal axis. In some embodiments, theFeed Ring equipment is connected to one or more pumps. In oneembodiment, the one or more pumps comprises a first pump (e.g., Q2 pump)and a second pump (e.g., RHO pump). In certain embodiments, the aqueoussolution comprising, for example, monomers (e.g., N-tris-hydroxymethylmethylacrylamide, diethylaminoethylacrylamide, andN,N-methylene-bis-acrylamide monomers) and/or gelatin is filtered andplaced under stirring. In some embodiments, the stirring is done at atemperature of about 25° C. to about 80° C., such as about 40° C. toabout 70° C. In some embodiments, the stirring is done at a temperatureof about 25° C., 40° C., 50° C., 55° C., 60° C., 65° C. or 70° C., orany range thereof. In a specific embodiment, the stirring is done at atemperature of about 60° C. In certain embodiments, the aqueous solutioncomprising microsphere monomers and gelatin is injected through thefirst pump into the Feed Ring equipment placed in a solution comprisingan oil, such as mineral oil (e.g., paraffin oil), and an emulsifier,such as a sorbitan sesquioleate (e.g., Arlacel 83; or similar product).In certain embodiments, the emulsifier (e.g., sorbitan sesquioleate) isat a concentration of from about 0.01 g/L to about 1 g/L, such as fromabout 0.02 g/L to about 0.5 g/L, or from about 0.03 g/L to about 0.3 g/Lin oil. In one embodiment, the emulsifier (e.g., sorbitan sesquioleate)is at a concentration of about 0.031 g/L in oil (e.g., 3.1 g (or 3 ml)of Arlacel in 4 liters of oil). In certain embodiments, the solutionfurther comprises N,N,N′,N′-tetramethylethylenediamine (TEMED).

In certain embodiments, an aqueous ammonium persulfate solution isprepared and injected through the second pump into the Feed Ringequipment placed in a solution comprising an emulsifier (e.g., sorbitansesquioleate). In one embodiment, the monomer/gelatin solution andammonium persulfate solution are simultaneously injected through thefirst pump and second pump, respectively, into the Feed Ring equipment.In some embodiments, the ammonium persulfate is at a concentration offrom about 0.01 g/mL to about 1 g/mL, such as from about 0.02 g/mL toabout 0.5 g/mL, or from about 0.03 g/mL to about 0.3 g/mL in water. Inone embodiment, the aqueous ammonium persulfate solution is at aconcentration of about 0.034 g/mL in water (e.g., 3.5 g of ammoniumpersulfate in 101.6 g of water). In some embodiments, the use of theFeed Ring process results in reduced oil encapsulation of microspheres,decreased numbers of small particles formed within larger particlesand/or reduced cracked microspheres.

2. Sonication

In certain embodiments, the microspheres provided herein are optionallysubjected to sonication (particularly ultrasonication). In specificembodiments, the microspheres are subjected to sonication (e.g.,ultrasonication) prior to, during or after gelatin crosslinking. In oneembodiment, microspheres provided herein are subjected to sonication(e.g., ultrasonication) prior to gelatin crosslinking. Sonication (e.g.,ultrasonication) can be used to break the interactions betweenmicrospheres and reduce the percentage of sticking microspheres presentin the microsphere population. The result of such a sonication step canresult in greater efficiency in producing non-sticking microspheres(i.e., reducing the amount of aggregated microspheres) and a morecost-effective way to produce microspheres by reducing the need for (ornumber of) one or more rounds of sieving in an effort to eliminatesticking microspheres from a given population of microspheres.

Any ultrasound generator can be used in combination with one or more ofthe processes or methods of making microspheres provided herein. Incertain embodiments, the sonication is performed using a sonicator, forexample, an ultrasonic bath, ultrasonic probe or ultrasonic processor.In one embodiment, the sonication is performed using an ultrasonic bath.In some embodiments, the sonication is performed one time. In otherembodiments, the sonication is performed more than one time. In certainembodiments, the sonication is performed at a frequency of from about 20kHz to about 200 kHz, such as from about 30 kHz to about 100 kHz, orfrom about 35 kHz to about 70 kHz. In some embodiments, the sonicationis performed at a frequency of about 35 kHz, 40 kHz, 45 kHz, 50 kHz, 55kHz, 60 kHz, 65 kHz, or about 70 kHz, or any range thereof. In oneembodiment, the sonication is performed at a frequency of about 35 kHz.In certain embodiments, the sonication is performed at a temperature offrom about 0° C. to about 80° C., such as from about 10° C. to about 50°C., from about 15° C. to about 35° C., from about 20° C. to about 25° C.(e.g., room temperature), or from about 0° C. to about 5° C. (e.g., onice), or any range thereof. In certain embodiments, the sonication isperformed at a temperature of about 0° C., 4° C., 5° C., 10° C., 25° C.or room temperature.

In certain embodiments, the sonication is done for from about 1 minuteto about 60 minutes, such as from about 5 minutes to about 40 minutes,or from about 10 minutes to about 15 minutes, or any range thereof. Insome embodiments, the sonication is done for about 5, 10, 15, or 20minutes. In one embodiment, the sonication is done for about 10 minutesor about 15 minutes. The time periods above can reflect a single,continuous sonication or multiple sonications.

In certain embodiments, the percentage of sticking (or aggregated)microspheres present after sonication is from about 0% to about 3%, fromabout 0% to about 2%, from about 0% to about 1.5%, from about 0% toabout 1%, or from about 0% to about 0.5%, or any range thereof. In someembodiments, the percentage of sticking spheres present in themicrospheres after sonication is about 0%, about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9% or about 1%. In certain embodiments, no broken microspheres areobserved after sonication.

In some embodiments, microspheres provided herein prepared usingsonication (e.g. ultrasonication) results in an improvement in theoverall manufacturing process of the microspheres (e.g., higher yieldand/or less expensive), which can lead to an improvement in the qualityof the microspheres (e.g., less broken and/or aggregated microspheres),a more consistent product (e.g., greater uniformity in size and/orshape), or a combination thereof, as compared to the same microsphereprepared without using ultrasonication. In specific embodiments,microspheres provided herein prepared using sonication (e.g.,ultrasonication) results in yields of greater than 30%, greater than35%, greater than 40%, greater than 50%, greater than 55%, greater than60% (or higher) of microspheres from a prepared batch (or population) ofmicrospheres, which comprise 5% or less, 4.5% or less, 3.5% or less, 3%or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% orless, 0.4% or less, 0.3% or less, 0.25% or less, 0.2% or less or 0.1% orless of aggregated microspheres.

In one embodiment, provided herein is a method of making microspherescomprising: (a) preparing an aqueous solution comprising: (i) aN-tris-hydroxymethyl methylacrylamide monomer, (ii) adiethylaminoethylacrylamide monomer, (iii) aN,N-methylene-bis-acrylamide monomer, and (iv) gelatin, wherein theN-tris-hydroxymethyl methylacrylamide monomer and/or thediethylaminoethylacrylamide monomer is an ultra-pure monomer; (b) addingthe aqueous solution to a liquid organic phase that has low miscibilityin water, before or while stirring; thereby producing microspherescomprising a copolymer comprising a N-tris-hydroxymethylmethylacrylamide monomer unit, a diethylaminoethylacrylamide monomerunit and a N,N-methylene-bis-acrylamide monomer unit; (c) optionallysubjecting the microspheres to ultrasonication; and (d) crosslinking thegelatin. In some embodiments, the aqueous solution is added through afeed ring to the liquid organic phase. In an embodiment, theN-tris-hydroxymethyl methylacrylamide monomer comprises less than 9% ofimpurities and/or the diethylaminoethylacrylamide monomer comprises lessthan 2% of impurities. In one embodiment, the microspheres areultra-pure microspheres. In another embodiments, the (i) the ultra-puremicrospheres comprise about 10% or less, 9% or less, 8% or less, 7% orless, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less or 1%or less of impurities, (ii) the ultra-pure microspheres comprise 90% ormore, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more,96% or more, 97% or more, 98% or more or 99% or more by weight of theN-tris-hydroxymethyl methylacrylamide, the diethylaminoethylacrylamide,the N,N-methylene-bis-acrylamide and the gelatin, or (iii) a combinationof (i) and (ii). In yet another embodiment, the ultrasonication is donein an ultrasonic bath. In certain embodiments, the method does notcomprise sieving the microspheres.

Also provided herein are microspheres prepared by any of the variousmethods provided herein. In one embodiment, the microspheres prepared bythese methods exhibit in a ¹H NMR spectrum, a first peak from about 3.5ppm to about 4 ppm, a second peak from about 3 ppm to about 3.5 ppm, anda third peak from about 1 ppm to about 1.5 ppm; and wherein (i.) theintegration ratio of the second peak to the first peak is from 0.50 toabout 0.65, (ii.) the integration ratio of the third peak to the firstpeak is from 0.61 to about 0.75, or (iii.) a combination thereof. In oneembodiment, the microspheres are uniform in size. In anther embodiment,the microspheres have a diameter from about 1 μm to about 2000 from 40μm to about 120 from about 100 μm to about 300 from about 300 μm toabout 500 from about 500 μm to about 700 from about 700 μm to about 900or from about 900 μm to about 1200 In some embodiments, less than 1% ofthe microspheres are aggregated microspheres.

D. Method of Use

In certain embodiments, the microspheres are flexible, but not fragile,such that they can easily pass into and through injection devices andsmall catheters without being permanently altered or broken, but themicrospheres are also resistant to the muscle contraction stressgenerated during and after the implantation process. In someembodiments, the microspheres are thermally stable which allows foreasy, convenient sterilization, and frozen storage.

Thus, the microspheres provided herein have a wide variety ofapplications. For example, the microspheres provided herein can be usedfor embolization, tissue engineering, tissue guided regeneration, invivo stem cell harvesting, culturing, or differentiation, delivery andsuspension of therapeutic materials in targeted human or animal tissuesand/or other applications.

Any of the various microspheres provided herein, or prepared by thevarious methods provided herein, can be used in any the variousembolization and disease management and treatment embodiments providedherein. In one embodiment, provided is a method of embolization in asubject, comprising administering to the subject microspheres providedherein. In another embodiment, provided herein is a method of managingor treating an angiogenesis-dependent disease in a subject, comprisingadministering to the subject microspheres provided herein. In oneembodiment, the angiogenesis-dependent disease is arteriovenousmalformation, uterine fibroid, or benign prostatic hyperplasia. In anembodiment, the angiogenesis-dependent disease is a cancer or tumor,such as a liver or prostate cancer or tumor.

1. Embolization

There are a number of clinical situations (e.g., bleeding, tumordevelopment) where it is desirable to reduce or abolish the blood supplyto an organ or region. As described in greater detail below, this can beaccomplished by injecting the microspheres or compositions into adesired blood vessel through a selectively positioned needle orcatheter, or under the guidance of an x-ray camera (e.g., afluoroscope). The microspheres or compositions travel via the bloodstream until it becomes wedged in the vasculature, thereby physically(or chemically) occluding the blood vessel. The reduced or abolishedblood flow to the selected area results in infarction (cell death due toan inadequate supply of oxygen and nutrients) or reduced blood loss froma damaged vessel.

Thus, in certain embodiments, provided herein is a method ofembolization in a subject or patient, comprising administering to thesubject a microsphere (microspheres) or a composition comprising themicrosphere(s). In one embodiment, provided are methods for embolizing ablood vessel, comprising administering to the vessel of a subject orpatient a therapeutically effective amount of the microspheres, suchthat the blood vessel is effectively occluded. In some embodiments,embolization can be accomplished in order to treat or prevent conditionsof excessive bleeding. Embolization therapy utilizing microspheres orcompositions provided herein can also be applied to a variety of otherclinical situations where it is desired to occlude blood vessels, forexample, for acute bleeding, vascular abnormalities, central nervoussystem disorders, and hypersplenism.

In the case of vascular malformations, such as AVM or arteriovenousfistulas, vascular occlusion enables the blood flow to the tissues to benormalized, aids in surgery, and limits the risk of hemorrhage. Inhemorrhagic processes, vascular occlusion produces a reduction of flow,which promotes cicatrization of the arterial opening(s).

Embolization can be used in the treatment of uterine fibroids,postpartum and/post-caesarian bleeding, post-surgical vaginal bleeding,the prevention and/or treatment of hemorrhage from ectopic pregnancy,prophilatically prior to myomectomy and in obstetrical patients at highrisk for bleeding, such as those patients with placenta previa, placentaaccreta, and twin fetal death. Embolization can also be used to stopuncontrolled bleeding, or to slow bleeding prior or during surgery, andfor sealing endoleaks into aneurysm sacs.

Any of the various diseases or disorders provided herein, or a symptomthereof, can be managed, treated or prevented according the methodsprovided herein.

Furthermore, depending on the pathological conditions treatedembolization can be carried out for temporary as well as permanentobjectives.

Embolization can also be used in combination with other clinicalprocedures, such as angiography. For example, a radiopaque contrastagent can be injected to the area to be embolized through, e.g., acatheter inserted percutaneously or by surgery into an artery or vein asan x-ray is taken. The blood vessel can then be embolized by refluxingmicrospheres provided herein through the catheter, until flow isobserved to cease. Occlusion can be confirmed by repeating theangiogram.

The microspheres provided herein can be administered to (or otherwisecontacted with) a blood vessel, a tissue or organ (e.g., heart, kidney,spinal cord, uterus, liver or pancreas) by means known in the art. Incertain embodiments, the microspheres are administered (e.g., byinjection) to a tissue or organ that has more than one blood supply, forexample the liver, lung, spine, spinal cord, uterus or pancreas. Incertain embodiments, the microspheres are administered to the heart,lung, nervous system, brain, lung, liver, uterus or pancreas of thepatient. In some embodiments, the microspheres are administered to oneor more blood vessels, veins or arteries comprised within the tissue ororgan. In certain embodiments, the microspheres provided herein are usedto counter ischemia in the target area, e.g., the area of administrationor injection, such as in or near a tissue or organ. In some embodimentsof the methods provided herein, the microspheres are administered to apatient by intraluminal administration or injection. In otherembodiments of the methods provided herein, the microspheres areadministered to a patient by intravascular administration or injection.

The microspheres can be delivered systemically or locally to the desiredblood vessel, tissue or organ. In some embodiments, the microspheres areadministered to a blood vessel, tissue or organ before, during or aftera surgery. In other embodiments, the microspheres are delivered to ablood vessel, tissue or organ using non-surgical methods, for example,either locally by direct injection into the target area, to a remotesite and allowed to passively circulate to the target site, or to aremote site and actively directed to the target site. Such non-surgicaldelivery methods include, for example, infusion or intravascular (e.g.,intravenous or intraarterial), intramuscular, intraperitoneal,intrathecal, intradermal or subcutaneous administration. In certainembodiments, angiography (e.g., selective angiography or superselectiveangiography) is used in conjunction with embolization to assess theblood supply to the tissue or organ. In such embodiments, an angiogramcan be taken prior to, during, or after embolization.

Diseases or disorders provided herein can be treated or otherwisemanaged by administering to the patient (e.g., a patient in needthereof) a therapeutically effective amount of the microspheres or acomposition provided herein.

In certain embodiments, administration is carried out by injection. Incertain embodiments, the microspheres are administered by a catheter. Inother embodiments, the microspheres are injected using a needle attachedto a syringe. In some embodiments, administration is into a bloodvessel. In other embodiments, administration is directly to the site ofaction, for example into a tumor mass, or into a cell, organ or tissuerequiring such treatment or management. In some embodiments, themicrospheres are administered in combination with a drug solution orother therapy, wherein the drug solution or other therapy isadministered prior, simultaneously or after the administration of themicrospheres.

It should be understood that the patients suitable for embolization withthe microspheres provided herein include humans and animals, includingmale and female infants, children, and adults, including the elderly. Ina specific embodiment, the patient is at risk for, or currentlyafflicted with, hepatocellular diseases, such as hepatitis or a livercancer or tumor, for example Caucasian or Asian (e.g., including, butnot limited to, people of Japanese heritage) human patients, 18 to 75years of age. In some embodiments, the patients are from 25 to 75 yearsof age, from 25 to 50 years of age, from 50 to 75 years of age, or from18 to 25 years of age. In one embodiment, the patient is less than 18years of age (e.g., from 1 to 5 years of age, from 5 to 10 years of age,from 10 to 15 years of age or from 15 to 18 years of age). In anotherembodiment, the patient is 75 years of age or older.

In some embodiments, the microspheres provided herein are used in thetreatment, management, or prevention of hepatocellular disease, or asymptom thereof, in a patient. In one embodiment, the patient isChild-Pugh class A. In another embodiment, the patient is Child-Pughclass B. In yet other embodiments, the patient is Child-Pugh class C.The Child-Pugh classification is well known in the art, see, e.g., Childand Turcotte (1964) Surgery and portal hypertension, In: The liver andportal hypertension (Edited by: Child CG). Philadelphia, Saunders 1964,50-64; which was later modified by Pugh et al. Transection of theesophagus in bleeding oesophageal varices (1973) Br. J. Surg.60:648-652. In some embodiments, the patient is infected with ahepatitis C virus (HCV).

Microspheres and compositions provided herein can also be in combinationwith drugs or other therapies. For example, the microspheres andcompositions can be used to treat or otherwise manage tumors or cancers(e.g., prostate or liver cancer), inflammatory diseases or otherdiseases associated with inflammation, or a symptom thereof. In otherembodiments, the microspheres and compositions provided herein can beused to treat or otherwise manage uterine fibroids, or a symptomthereof. In other embodiments, the microspheres and compositionsprovided herein can be used to treat or otherwise manage a vascularmalformation, such as an AVM, or a symptom thereof. In yet otherembodiments, the microspheres and compositions provided herein can beused to treat or otherwise manage a prostate disease, such as a benignprostate hyperplasia, or a symptom thereof.

In certain embodiments, a drug or other therapy is administeredconcurrently to the subject in combination with the microspheresprovided herein. In some embodiments, a drug or other therapy isadministered to the subject prior to administration of microspheres. Incertain embodiments, a drug or other therapy is administered from about1 minute to about 60 minutes prior to administration of microspheres. Insome embodiments, a drug or other therapy is administered to the subjectwithin about 1 minute, about 5 minutes, about 10 minutes, about 15minutes, about 20 minutes, about 30 minutes, about 45 minutes or about 1hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about12 hours, about 18 hours, about 20 hours or about 24 hours ofadministration of microspheres. In yet other embodiments, a drug orother therapy is administered concurrently with microspheres. In certainembodiments, microspheres are administered to the subject prior toadministration of a drug or other therapy. In certain embodiments,microspheres are administered between about 1 minute and about 60minutes prior to administration of a drug or other therapy. In someembodiments, microspheres are administered to the subject within about 1minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20minutes, about 30 minutes, about 45 minutes or about 1 hour, about 2hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours,about 18 hours, about 20 hours or about 24 hours of administration of adrug or other therapy.

2. Angiogenesis-Dependent Diseases

Angiogenesis-dependent diseases (i.e., those diseases which require orinduce vascular growth) represent a significant portion of all diseasesfor which medical treatment is sought. Such diseases include, forexample, cancers or tumors (e.g., liver cancers or tumors, or prostatecancer or tumors) and non-tumorigenic angiogenesis-dependent diseases.

In certain embodiments, provided herein are microspheres, compositionsand methods suitable for treating or otherwise managingangiogenesis-dependent diseases, including tumors or other cancers,non-tumorigenic angiogenesis-dependent diseases, or pain, such as painrelated to the presence of a tumor or other cancer, or a symptomthereof. In one embodiment, methods are provided for managing ortreating an angiogenesis-dependent disease in a subject, comprisingadministering to the subject a microsphere (microspheres) or acomposition comprising the microsphere(s). In specific embodiments,methods are provided for managing or treating an angiogenesis-dependentdiseases in a subject comprising, for example, administering to thesubject a microsphere (microspheres) or a composition comprising themicrosphere(s).

In addition to cancer, numerous other non-tumorigenicangiogenesis-dependent diseases which are characterized by the abnormalgrowth of blood vessels can also be treated, either via down-regulationor up-regulation, or otherwise managed with the microspheres orcompositions provided herein. Representative examples of suchnontumorigenic angiogenesis-dependent diseases include, withoutlimitation, hypertrophic scars and keloids, proliferative diabeticretinopathy, rheumatoid arthritis, arteriovenous malformation (AVM),lymphangitic malformations, venous malformations, atheroscleroticplaques, delayed wound healing, hemophilic joints, nonunion fracturesKlippel Trenaunay Syndrome, Parkes Weber Syndrome, Osler-Weber-RenduSyndrone, Blue Rubber Bleb Syndrome, cutnaoues and subcutaneous nevi,hemangiomas, leiomyomata, adenomas, hamartomas, psoriasis, pyogenicgranuloma, scleroderma, tracoma, menorrhagia, vascular adhesions, benignprostatic hyperplasia (BPH) and uterine fibroids.

a. Cancers or Tumors

In specific embodiments, methods are provided for managing or treating acancer or tumor (e.g., a hypervascularized cancer or tumor) in a subjectcomprising, for example, administering to the subject a microsphere(microspheres) or a composition comprising the microsphere(s). Suchcancers include, without limitation (both anatomically and by primaryneoplastic site), liver, ovarian, breast, kidney, lung, pancreatic,thyroid, prostate, uterine, skin cancer, head and neck tumors, breasttumors, brain, bone, soft tissues (such as sarcoma, lipoma, malignanyfibrous histiocytoma), blood (such as lymphoma), Kaposi's sarcoma, andsuperficial forms of bladder cancer. In certain embodiments, the methodof treatment or management can be the result of localized (or systemic)drug delivery in combination with embolic effects of the microspheres(e.g., TACE).

Other diseases, and symptoms thereof, contemplated for management andtreatment with the compositions and methods provided herein include, forexample, without limitation, tumors associated with the liver, kidney,acute lymphoblastic leukemia, acute myeloid leukemia, Ewing's sarcoma,gestational trophoblastic carcinoma, Hodgkin's disease, non-Hodgkin'slymphoma, Burkitt's lymphoma diffuse large cell lymphoma, follicularmixed lymphoma, lymphoblastic lymphoma, rhabdomyosarcoma, testicularcarcinoma, wilms's tumor, anal carcinoma, bladder carcinoma, breastcarcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia,hairy cell leukemia, head and neck carcinoma, meningioma, neurofibrosoma, angio fibrosoma, lung (small cell) carcinoma, multiplemyeloma, Non-Hodgkin's lymphoma, follicular lymphoma, ovarian carcinoma,brain tumors (astrocytoma), cervical carcinoma, colorectal carcinoma,hepatocellular carcinoma, human large hepatocellular carcinoma, Kaposi'ssarcoma, lung (non-small-cell) carcinoma, melanoma, pancreaticcarcinoma, prostate carcinoma, soft tissue sarcoma, breast carcinoma,colorectal carcinoma (stage II), bone tumors, osteogenic sarcoma,ovarian carcinoma, testicular carcinoma, or combinations thereof.

Embolization therapy using the microspheres or compositions providedherein can be utilized in at least three principal ways to assist in themanagement of neoplasms: (1) definitive treatment of tumors (usuallybenign); (2) for preoperative embolization; and (3) for palliativeembolization. Briefly, benign tumors can sometimes be successfullytreated by embolization therapy alone. Examples of such tumors includesimple tumors of vascular origin (e.g., haemangiomas), endocrine tumorssuch as parathyroid adenomas, and benign bone tumors.

For other tumors, (e.g., renal adenocarcinoma), preoperativeembolization can be employed hours or days before surgical resection inorder to reduce operative blood loss, shorten the duration of theoperation, and reduce the risk of dissemination of viable malignantcells by surgical manipulation of the tumor. Many tumors can besuccessfully embolized preoperatively, including for examplenasopharyngeal tumors, glomus jugular tumors, meningiomas,chemodectomas, and vagal neuromas.

Embolization using the microspheres or compositions can also be utilizedas a primary mode of treatment for inoperable malignancies, in order toextend the survival time of patients with advanced disease. Embolizationcan produce a marked improvement in the quality of life of patients withmalignant tumors by alleviating unpleasant symptoms such as bleeding,venous obstruction and tracheal compression. The benefits frompalliative tumor embolization, in certain embodiments, can be seen inpatients suffering from the humoral effects of malignant endocrinetumors, wherein metastases from carcinoid tumors and other endocrineneoplasms such as insulinomas and glucagonomas can be slow growing, andyet still cause great distress by virtue of the endocrine syndromeswhich they produce. In certain embodiments, embolization therapy canalso be used during surgery to remove a tumor or vascular mass orcancerous organ, or to prevent or ameliorate metastasis.

Chemoembolization is a combination of chemotherapy and embolization orembolotherapy, used typically to treat cancer. Similarly,radioembolization is a combination of radiation therapy and embolizationor embolotherapy. In certain embodiments, the microspheres providedherein can be injected to a target area as a standalone therapy or forthe purposes of interspersion between terminal therapeutic microspheresto allow for gradual migration of the microspheres into tumor bloodsupply, while providing continued perfusion/blood flow into targetedtumor. The addition of chemotherapeutics to the microsphere matrix canincrease the efficacy of the therapy by improving the timing of exposureof therapy with the terminal embolic effect of microspheres.

A wide variety of cancers or tumors may be embolized utilizing amicrosphere composition provided herein. Briefly, tumors are typicallydivided into two classes: benign and malignant. In a benign tumor, thecells can retain their differentiated features and do not divide in acompletely uncontrolled manner. In addition, the tumor is localized andnon-metastatic. In a malignant tumor, the cells can becomeundifferentiated, do not respond to the body's growth and hormonalsignals, and multiply in an uncontrolled manner; the tumor is invasiveand capable of spreading to distant sites (metastasizing). Bother benignand malignant tumors can be embolized, treated, managed, prevented orameliorated using the microspheres provided herein.

In certain embodiments, also provided herein are methods of managing ortreating secondary tumors (e.g., secondary hepatic tumors) using themicrospheres or compositions provided herein. A secondary tumor, ormetastasis, is a tumor which originated elsewhere in the body but hassubsequently spread to a distant organ. The common routes for metastasisare direct growth into adjacent structures, spread through the vascularor lymphatic systems, and tracking along tissue planes and body spaces(peritoneal fluid, cerebrospinal fluid, etc.).

In other embodiments of the methods provided herein, embolizationtherapy may be used during surgery to remove a tumor or vascular mass orcancerous organ. Additionally, therapeutic embolization therapy can beused to treat, manage, prevent or ameliorate metastasis.

In certain embodiments, blood vessels which nourish a tumor aredeliberately blocked by injection of an embolic material into thevessel. Notably, in the case of tumors, vascular occlusion methodsprovided herein can be used to suppress pain, limit blood loss on thesurgical intervention to follow embolization, or even bring on a tumoralnecrosis and avoid an operation.

i. Liver Cancers or Tumors

In certain embodiments, liver cancers or tumors can be treated ormanaged utilizing the methods comprising administering the microspheresor compositions to the subject. Representative examples of benignhepatic tumors include hepatocellular adenoma, cavernous haemangioma,and focal nodular hyperplasia. Other benign tumors, which are more rareand often do not have clinical manifestations, can also be treated.These include bile duct adenomas, bile duct cystadenomas, fibromas,lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, and nodularregenerative hyperplasia.

Malignant hepatic tumors can be subdivided into two categories: primaryand secondary. Primary tumors arise directly from the tissue in whichthey are found. Thus, a primary liver tumor is derived originally fromthe cells which make up the liver tissue (such as hepatocytes andbiliary cells). Representative examples of primary hepatic malignanciesinclude hepatocellularcarcinoma, cholangiocarcinoma, angiosarcoma,cystadenocarcinoma, squamous cell carcinoma, and hepatoblastoma. Hepaticmalignancies, or symptoms thereof, can be treated or otherwise managed,for example, using the compositions and methods provided herein.

Arterial embolization can be done, for example, by injectingmicrospheres through a small tube, or catheter, threaded into thehepatic artery. For example, a catheter can be inserted via the femoralor brachial artery and advanced into the hepatic artery by steering itthrough the arterial system under fluoroscopic guidance. The cathetercan be advanced into the hepatic arterial tree as far as necessary toallow complete blockage of the blood vessels supplying the tumor(s),while sparing as many of the arterial branches supplying normalstructures as possible. This can be, for example, a segmental branch ofthe hepatic artery, but it could be that the entire hepatic arterydistal to the origin of the gastroduodenal artery, or even multipleseparate arteries, will need to be blocked depending on the extent oftumor and its individual blood supply. Once the desired catheterposition is achieved, the artery can be embolized by injectinganti-angiogenic therapeutic compositions (e.g., microspheres providedherein and optionally one or more additional therapies) through thearterial catheter until flow in the artery to be blocked ceases, such asafter observation for 5 minutes. Occlusion of the artery can beconfirmed by injecting radiopaque contrast through the catheter anddemonstrating by fluoroscopy or x-ray film that the vessel whichpreviously filled with contrast no longer does so. The same procedurecan be repeated with each feeding artery to be occluded.

The hepatic artery is the main source of blood for most liver tumors,and thus, microspheres can block the flow of blood to the tumor,depriving it of the nutrients and oxygen it needs to survive. In asimilar manner, arterial embolization can be accomplished in a varietyof other conditions, including for example, without limitation, foracute bleeding, vascular abnormalities, central nervous systemdisorders, and hypersplenism. In certain embodiments, transarterialchemoembolization (TACE) and transarterial embolization (TAE) can beperformed to treat liver cancers or tumors. TACE is a combinationtherapy of TAE and regional chemotherapy, which refers to aninterventional radiology procedure involving gaining percutaneous accessto the hepatic artery, usually by puncturing the common femoral arteryin the right groin and passing a catheter through the abdominal aorta,through the celiac axis and common hepatic artery, into the properhepatic artery (which supplies the liver).

Selective arterial obstruction can induce ischemic tumor necrosis whileminimizing damage to the liver tissue. The blood supply to the livertissue is still maintained by dominant blood flow from the portal veinminimizing damage to the liver. In addition, chemotherapeutic agentsconcomitantly administered remain in a tumor for a longer period at ahigher concentration. The embolotherapy interrupts the arterial bloodflow to a tumor and prevents washout of the injected chemotherapeuticagents from a tumor.

TACE can derive its beneficial effect in two ways. Since most tumors aresupplied by the hepatic artery, arterial embolization interrupts theirblood supply and postpones growth until replaced by neovascularity.Further, focused administration of chemotherapy allows a higher dose tothe tissue while simultaneously reducing systemic exposure, which istypically the dose limiting factor. This effect is potentiated by thefact that the chemotherapeutic drug is not washed out from the tumor bedafter embolization. Thus, the combination of embolotherapy and regionalchemotherapy has synergistic, anti-tumor effects with a high objectiveresponse rate. Another added benefit is that the use of combinationtherapy results in lower systemic drug levels and therefore lesstoxicity.

In certain embodiments, also provided herein are methods of managing ortreating secondary hepatic tumors) using the microspheres orcompositions provided herein. Secondary hepatic tumors are one of themost common causes of death in the cancer patient and are by far andaway the most common form of liver tumor. Although virtually anymalignancy can metastasize to the liver, tumors which are most likely tospread to the liver include: cancer of the stomach, colon, and pancreas;melanoma; tumors of the lung, oropharynx, and bladder; Hodgkin's andnon-Hodgkin's lymphoma; tumors of the breast, ovary, and prostate. Eachone of the above-named primary tumors has numerous different tumor typeswhich can be treated by arterial embolization (for example, withoutlimitation, there are reportedly over 32 different types of ovariancancer).

ii. Prostate Cancers or Tumors

In certain embodiments, methods are provided for managing or treatingprostate cancers or tumors, or a symptom thereof, using microspheres orcompositions provided herein.

In some embodiments, the microspheres or compositions are administeredto an area surrounding the prostate, such as, the prostatic artery. Forexample without limitation, the microspheres or compositions can bedelivered to a blood vessel that nourishes the prostate cancer.

The administration of microspheres or compositions can be conducted viaa syringe, a catheter, a needle and other means for injecting orinfusing. The syringe, the catheter, the needle or the like can beinserted into a vein or an artery, for example, the femoral artery orthe inferior vesicle artery.

In certain embodiments, a syringe, a catheter, or a needle is advancedinto, for example, the ostium of the prostate arteries and, in oneembodiment, advanced as far as necessary to allow complete blockage ofthe blood vessels supplying a prostate cancer, while sparing as many ofthe arterial branches supplying normal structures as possible.

In some embodiments of the methods provided herein, aniography of thearea to be embolized is performed prior to embolization. The bloodvessel is then embolized by refluxing an embolic material providedherein through a previously placed catheter, until flow is observed tocease. The catheter can be inserted either percutaneously or by surgery.Occlusion can be confirmed by repeating the angiogram.

b. Arteriovenous Malformation

In further specific embodiments, methods are provided for managing ortreating arteriovenous malformation or a symptom thereof in a subjectcomprising, for example, administering to the subject a microsphere(microspheres) or a composition comprising the microsphere(s) to occludearteries or veins to correct the arteriovenous malformation. In oneembodiment, the arteriovenous malformation can be treated by inserting acatheter via the femoral or brachial artery, and advancing it into thefeeding artery under fluoroscopic guidance. The catheter can be advancedas far as necessary to allow complete blockage of the blood vesselssupplying the vascular malformation, while sparing as many of thearterial branches supplying normal structures as possible (ideally thiswill be a single artery, but most often multiple separate arteries mayneed to be occluded, depending on the extent of the vascularmalformation and its individual blood supply). Once the desired catheterposition is achieved, each artery can be embolized utilizing themicrospheres or compositions provided herein.

c. Uterine Fibroids

In further specific embodiments, methods are provided for managing ortreating uterine fibroids or a symptom thereof, for example, by usinguterine fibroid embolization (UFE) or uterine artery embolization (UAE).The cause of uterine fibroids is unknown. However, they commonly causeheavy menstrual bleeding, pain in the pelvic region, and pressure on thebladder or bowel.

In certain embodiments, embolization (such as UFE) using themicrospheres and compositions provided herein can be accomplished inorder to treat conditions of excessive bleeding, including excessivebleeding associated with uterine fibroids. For example, menorrhagia(excessive bleeding with menstruation) can be readily treated byembolization of uterine arteries (e.g., branches of the internal iliacarteries bilaterally). In certain embodiments, the compositions andmethods provided herein are used to manage or treat symptoms of uterinefibroids, such as heavy menstrual bleeding, pelvic pain or pressureand/or urinary dysfunction.

In some embodiments, a catheter may be inserted via the femoral orbrachial artery, and advanced into each uterine artery by steering itthrough the arterial system under the guidance of an x-ray camera (e.g.,a fluoroscope). In certain embodiments, the catheter can be advanced asfar as necessary to allow complete blockage of the blood vessels to theuterus, while sparing as many arterial branches that arise from theuterine artery and supply normal structures as possible. In certainembodiments, a single uterine artery on each side may be embolized, butoccasionally multiple separate arteries may need to be blocked dependingon the individual blood supply. Once the desired catheter position isachieved, each artery can be embolized by administration of themicrospheres and compositions as described above. The administeredmicrospheres block the arteries that provide blood flow, causing thefibroids to shrink, and reliving the symptoms of women with fibroids. Incertain embodiments, UAE can also be used to stop severe pelvic bleedingcaused, for example, by trauma, malignant gynecological tumors orhemorrhage after childbirth.

D. Benign Prostatic Hyperplasia

In further specific embodiments, methods are provided for managing ortreating benign prostatic hyperplasia (BPH) or a symptom thereof. Themost frequent obstructive urinary symptoms are hesitancy, decreasedurinary stream, intermittency, sensation of incomplete emptying,nocturia, frequency and urgency.

In certain embodiments, the management or treatment of BPH can beaccomplished by embolization such as prostatic artery embolization (PAE)or transcatheter arterial embolization (TAE) using the microspheres andcompositions provided herein.

In some embodiments, a catheter (e.g., a microcatheter) can be insertedinto the right and/or left inferior vesicle arteries under the guidanceof an x-ray camera (e.g., a fluoroscope). In certain embodiments, thecatheter can be advanced as far as necessary to allow complete blockageof the blood vessels to the prostate, while sparing as many arterialbranches that arise from the prostate artery and supply normalstructures as possible.

In certain embodiments, angiography (e.g., initial pelvic angiography orselective digital subtraction angiography) can be used in conjunctionwith embolization to evaluate the iliac vessels and prostate arteriesduring the arterial and late phases, or to assess the blood supply tothe prostate. Once the desired catheter position is achieved, eachartery can be embolized by administration of the microspheres andcompositions as described above. The administered microspheres can blockthe arteries that provide blood flow, reducing the prostate size, andreliving the symptoms of BPH. In certain embodiments, embolization canalso be used to control massive hemorrhage after prostatectomy orprostate biopsy.

3. Diagnostic Imaging

As discussed above, the microspheres provide herein may be used inconnection with diagnostic imaging, therapeutic imaging and therapeuticdrug delivery, including, for example, ultrasound (US), magneticresonance imaging (I), nuclear magnetic resonance (NMR), computedtomography (CT), electron spin resonance (ESR), nuclear medical imaging,optical imaging, elastography, drug delivery with ultrasound,radiofrequency (RF) and microwave laser.

In certain embodiments, the microspheres provided herein arefluoroscopically visible. That is, in some embodiments, the microspheresare loaded with, associated with, or otherwise contain one or moresuitable contrast agents, such as an ionic or non-ionic contrast agent.

In some embodiments, the microspheres provided herein comprise anon-ionic contrast agent. The contrast agent can be loaded on themicrosphere, associated with the microsphere, absorbed by, adsorbed byor otherwise contained in or on the microsphere. Alternatively, thecontrast agent is a carrier solution for the microsphere. In specificembodiments, the contrast agent, such as a non-ionic contrast agent, isloaded within the microsphere (e.g., by mixing of otherwise contactingthe contrast agent with the microspheres). In other embodiments, themicrospheres do not comprise a contrast agent, such as a non-ioniccontrast agent.

The non-ionic contrast agents can be an X-ray, CT, MM contrast agent, ora combination thereof. The contrast agent can be paramagnetic orsuperparamagnetic. In some embodiments, the contrast agent is an X-raycontrast agent (also referred to as fluoroscopic agent or radio-opaque)or a CT contrast agent. In certain embodiments, the agent containsiodine. The non-ionic contrast agents can be monomeric, dimeric, orpolymeric.

Examples of non-ionic contrast agents include, without limitation,metrizamide, iopamidol (Isovue™ or Iopamiron™), iodixanol (Visipaque™),iohexol (Omnipaque™) iopromide (Ultravist™), iobtiridol, iomeprol,iopentol, iopamiron, ioxilan, iotrolan, gadodiamide, gadoteridol,iotrol, ioversol (Optiray™) or combinations thereof. In certainembodiments, the contrast agent is iopamidol. In specific embodiments,non-ionic contrast agent isiodixanol, iohexal, iopromide, or ioversol.In another embodiment, the non-ionic contrast agent is gadodiamide orgadoteridol.

Further examples of suitable contrast agents for use in combination withthe present stabilizing materials include stable free radicals, such as,stable nitroxides, as well as compounds comprising transition,lanthanide and actinide elements, which may, if desired, be in the formof a salt or may be covalently or non-covalently bound to complexingagents, including lipophilic derivatives thereof, or to proteinaceousmacromolecules. The transition, lanthanide and actinide elements caninclude, for example, Gd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III),Co(II), Er(II), Ni(II), Eu(III) and Dy(III). In some embodiments, theelements are Gd(III), Mn(II), Cu(II), Fe(II), Fe(III), Eu(III) andDy(III). The foregoing elements may be in the form of a salt, includinginorganic salts, such as a manganese salt, for example, manganesechloride, manganese carbonate, manganese acetate, and organic salts,such as manganese gluconate and manganese hydroxylapatite. Otherexemplary salts include salts of iron, such as iron sulfides, and ferricsalts, such as ferric chloride.

The above elements may also be bound, for example, through covalent ornoncovalent association, to complexing agents, including lipophilicderivatives thereof, or to proteinaceous macromolecules. Complexingagents can include, for example, diethylenetriaminepentaacetic acid(DTPA), ethylenediaminetetraacetic acid (EDTA),1,4,7,10-tetraazacyclododecane-N,N′,N′,N′″-tetraacetic acid (DOTA),1,4,7,10-tetraazacyclododecane-N,N′,N″-triacetic acid (DOTA),3,6,9-triaza-12-oxa-3,6,9-tricarboxymethylene-10-carboxy-13-phenyltridecanoicacid (B-19036), hydroxybenzylethylenediamine diacetic acid (HBED),N,N′-bis(pyridoxyl-5-phosphate)ethylene diamine, N,N′-diacetate (DPDP),1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA),1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA),kryptands (macrocyclic complexes), and desferrioxamine. In someembodiments, the complexing agents are EDTA, DTPA, DOTA, DO3A andkryptands, such as DTPA. Lipophilic complexes can include alkylatedderivatives of the complexing agents EDTA, DOTA, for example,N,N′-bis-(carboxydecylamidomethyl-N-2,3-dihydroxypropyl)ethylenediamine-N,N′-diacetate (EDTA-DDP);N,N′-bis-(carboxyoctadecylamidomethyl-N-2,3-dihydroxypropyl)ethylenediamine-N,N′-diacetate(EDTA-ODP); andN,N′-Bis(carboxy-laurylamidomethyl-N-2,3-dihydroxypropyl)ethylenediamine-N,N′-diacetate(EDTA-LDP); including those described in U.S. Pat. No. 5,312,617, thedisclosure of which is hereby incorporated herein by reference in itsentirety. Proteinaceous macromolecules can include, for example,albumin, collagen, polyarginine, polylysine, polyhistidine, γ-globulinand β-globulin. In certain embodiments, the proteinaceous macromoleculeis an albumin, polyarginine, polylysine or polyhistidine. Suitablecomplexes therefore include Mn(II)-DTPA, Mn(II)-EDTA, Mn(II)-DOTA,Mn(II)-DO3A, Mn(II)-kryptands, Gd(III)-DTPA, Gd(III)-DOTA, Gd(III)-DO3A,Gd(III)-kryptands, Cr(III)-EDTA, Cu(II)-EDTA, or iron-desferrioxamine.In specific embodiments, the complexes are Mn(II)-DTPA or Gd(III)-DTPA.

Nitroxides are paramagnetic contrast agents which increase both T1 andT2 relaxation rates on MRI by virtue of the presence of an unpairedelectron in the nitroxide molecule. As known to one of ordinary skill inthe art, the paramagnetic effectiveness of a given compound as an Millcontrast agent may be related, at least in part, to the number ofunpaired electrons in the paramagnetic nucleus or molecule, andspecifically, to the square of the number of unpaired electrons. Forexample, gadolinium has seven unpaired electrons whereas a nitroxidemolecule has one unpaired electron. Thus, gadolinium is generally a muchstronger MM contrast agent than a nitroxide. However, effectivecorrelation time, another important parameter for assessing theeffectiveness of contrast agents, confers potential increased relaxivityto the nitroxides. When the tumbling rate is slowed, for example, byattaching the paramagnetic contrast to a large molecule, it will tumblemore slowly and thereby more effectively transfer energy to hastenrelaxation of the water protons. In gadolinium, however, the electronspin relaxation time is rapid and will limit the extent to which slowrotational correlation times can increase relaxivity. For nitroxides,however, the electron spin correlation times are more favorable andtremendous increases in relaxivity may be attained by slowing therotational correlation time of these molecules. Nitroxides can bedesigned to coat the perimeters of the microspheres, for example, bymaking alkyl derivatives thereof, the resulting correlation times can beoptimized. Moreover, the resulting contrast medium may be viewed as amagnetic sphere, a geometric configuration which maximizes relaxivity.

Exemplary superparamagnetic contrast agents suitable for use in thecompositions herein include metal oxides and sulfides which experience amagnetic domain, ferro- or ferrimagnetic compounds, such as pure iron,magnetic iron oxide, such as magnetite, γ-Fe₂O₃, Fe₃O₄, manganeseferrite, cobalt ferrite and nickel ferrite. MR whole body imaging maythen be employed to rapidly screen the body, for example, forthrombosis, and ultrasound may be applied, if desired, to aid inthrombolysis.

The contrast agents, such as the paramagnetic and superparamagneticcontrast agents described above, may be employed as a component withinthe microspheres and/or stabilizing materials. With respect to vesicles,the contrast agents may be entrapped within the internal void thereof,administered as a solution with the microspheres, incorporated with anyadditional stabilizing materials, or coated onto the surface or membraneof the vesicle. Mixtures of any one or more of the paramagnetic agentsand/or superparamagnetic agents in the present compositions may be used.The paramagnetic and superparamagnetic agents may also be coadministeredseparately, if desired.

If desired, the paramagnetic or superparamagnetic agents may bedelivered as alkylated or other derivatives incorporated into thecompositions, especially the lipidic walls of the microspheres. Inparticular, the nitroxides 2,2,5,5-tetramethyl-1-pyrrolidinyloxy, freeradical and 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical, can formadducts with long chain fatty acids at the positions of the ring whichare not occupied by the methyl groups via a variety of linkages,including, for example, an acetyloxy linkage.

The microspheres provided herein may serve not only as effectivecarriers of the superparamagnetic agents described above, but also mayimprove the effect of the susceptibility contrast agents.Superparamagnetic contrast agents include metal oxides, particularlyiron oxides but including manganese oxides, and as iron oxides,containing varying amounts of manganese, cobalt and nickel whichexperience a magnetic domain. These agents are nano or microspheres andhave very high bulk susceptibilities and transverse relaxation rates.The larger particles, for example, particles having diameters of about100 nm, have much higher R₂ relaxivities as compared to R₁ relaxivities.The smaller particles, for example, particles having diameters of about10 to about 15 nm, have somewhat lower R₂ relaxivities, but much morebalanced R₁ and R₂ values. Much smaller particles, for example,monocrystalline iron oxide particles having diameters of about 3 toabout 5 nm, have lower R₂ relaxivities, but probably the most balancedR₁ and R₂ relaxation rates. Ferritin can also be formulated toencapsulate a core of very high relaxation rate superparamagnetic iron.

The iron oxides may simply be incorporated into the stabilizingmaterials and/or microspheres. In specific embodiments, the iron oxidesmay be incorporated into the walls of the microspheres, for example, bybeing adsorbed onto the surfaces of the microspheres, or entrappedwithin the interior of the microspheres.

E. Kits

Also provided herein are pharmaceutical packs and kits comprising one ormore containers filled with one or more of the ingredients of thecompositions provided herein. The kits can comprise, for example,microspheres and one or more additional components, wherein one, two,three or more of the components can be in one, two, three or more vials.In certain embodiments the microspheres are provided in the form of adry powder. In other embodiments, the microspheres are provided in abiocompatible carrier, for example as an emulsion or suspension.

In certain embodiments, the container is a syringe (e.g., apolycarbonate, polypropylene, or cyclic olefin polymer (COP) syringe).In specific embodiments, the syringe has low moisture loss, which canresult in an increased shelf-life (e.g., 2 to 3 years or longer) forpre-filled syringe embodiments of the kits provided herein. In aspecific embodiment, microspheres provided herein are contained within asterile syringe, such as a sterile pre-filled syringe (e.g., a 20 ccsyringe), that is optionally provided in a peel-away pouch. In certainembodiments, the syringe comprises about 1 ml, 2 ml, 3 ml or 4 ml of themicrospheres in a pharmaceutically acceptable carrier, such as saline(e.g., a non-pyrogenic or pyrogen-free, sterile physiological saline).

In other embodiments, the microspheres provided herein are containedwithin a vial. In specific embodiments, the vial is a glass vial with ascrew-off cap (e.g., a 5 ml glass vial), that is optionally packaged ina peel-away pack comprising one or more additional vials. In specificembodiments, the vial comprises 1 ml or 2 ml of the microspheres in apharmaceutically acceptable carrier, such as saline (e.g., anon-pyrogenic or pyrogen-free, sterile physiological saline).

Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for patient (e.g.,human or other mammal) administration. Also associated with suchcontainer(s) can be instructions for use. The reagents of any of themethods described herein can also be included as components of a kit.

In one kit format, the microspheres provided herein are present in aliquid, physiologically compatible solution in one vial. In another kitformat, the microspheres of the provided herein are present in dry formin one vial. In certain kit formats comprising multiple components inmultiple vials, the contents of the vials can be mixed together prior toor concurrently with administration. In some embodiments, themicrospheres are suspended in a suitable liquid prior to administration,or optionally a second vial is provided, which contains the injectablesolution and the contents of both vials are combined prior toadministration or concurrently with administration.

Finally, in another kit format the microspheres provided herein arepresent in one vial and a second vial contains a pharmaceuticallyacceptable solution comprising the contrast agent. The microspheres canthen be mixed together with the contrast agent, for example, prior to orconcurrently with administration.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES

The practice of the invention employs, unless otherwise indicated,conventional techniques in molecular biology, organic chemistry,biochemistry and related fields within the skill of the art. Thesetechniques are described in the references cited herein and are fullyexplained in the literature. See, e.g., Maniatis et al. (1982) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press;Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons (1987 and annual updates); CurrentProtocols in Immunology, John Wiley & Sons (1987 and annual updates).

Example 1 Synthesis of an Ultra-Pure Diethylaminoethylacrylamide Monomer

An ultra-pure diethylaminoethylacrylamide monomer was synthesizedaccording to the following scheme:

Briefly, acryloyl chloride was dissolved in CH₂Cl₂ and cooled to 0° C.N,N-Diethylethylenediamine dissolved in CH₂Cl₂ was added drop wise atT_(i)=0-5° C. followed by NaOH (˜15% aq. solution). The reaction mixturewas stirred for 15 min and the layers were separated. The organic layerwas washed with water and NaHCO₃ solution and dried over MgSO₄. Thesuspension was filtered and concentrated under reduced pressure andstabilized by BHT.

Example 2 HPLC Analysis of a Diethylaminoethylacrylamide Monomer

First, the standard solutions were prepared as follows, with MBA chosenas an internal reference control:

Stock Solutions

DEAE stock solution: approximately 200 mg (or 250 mg) of DEAE wasweighed in a 200 mL (or 250 mL) reference flask and dissolved in purewater (Purelab). The pH of the solution was adjusted to 2 by 1N HCl. Thesolution was adjusted with water to a concentration of 1000 ppm (mg/L).

MBA stock solution: approximately 200 mg (or 250 mg) of MBA was weighedin a 200 mL (or 250 mL) reference flask and dissolved in pure water. Thesolution was then subjected to ultrasonication for 15 min and let returnto room temperature. The solution was adjusted to a concentration of1000 ppm (mg/L).

Intermediate solution: an intermediate solution was prepared by diluting5 mL of the 1000 ppm solution with water to a final volume of 50 mL.Also prepared were 20, 40, 60, 80, 100 ppm.

The injected solutions are listed in Table 1. The exact concentrationsof the solutions were noted.

TABLE 1 Injected solutions DEAE solution DEAE solution MBA solutionTotal Solution concentration volume volume volume Standard 1 20 4 1 5Standard 2 40 4 1 5 Standard 3 60 4 1 5 Standard 4 80 4 1 5 Standard 5100 4 1 5

The samples to be analyzed were prepared as follows:

Stock solutions: approximately 200 mg (or 250 mg) of DEAE was weighed ina 200 mL (or 250 mL) reference flask and dissolved in pure water(Purelab). The pH of the solution was adjusted to 2 by 1N HCl. Thesolution was adjusted with water to a concentration of 1000 ppm (mg/L).Three stock solutions were prepared and tested.

Intermediate solution: an intermediate solution was prepared by diluting5 ml of the 1000 ppm solution with water to a final volume of 50 mL.

Sample solutions: sample solutions were prepared by diluting theintermediate solutions to approximately 60 ppm (at least 5 mL for sampleinjection). The intermediate solution (100 ppm) was also tested. Theexact concentrations were recorded.

The solutions (standards and samples) were then transferred to the HPLCvials on the support of the injector for analysis. The settings of theHPLC are listed in Table 2.

TABLE 2 HPLC settings Parameters Settings Injection Volume 20 μL OvenTemperature 30° C. Mobile phase Methanol/water (0.1% TFA) 10/90 Analysistime 6 min Wavelength 230 nm Column C18; 100 × 4.6 mm, 5 μm. Flow 1mL/min

The results of HPLC analysis are shown m FIG. 1A, which depicts thesensitivities of purity observed using a bromination reaction (rightbars, as provided by manufacturer BioSepra) and HPLC (left bars) forgiven lots of DEAE monomer (T209, U088 and U089) obtained from BioSepra.

Example 3 Bromine Test of a Diethylaminoethylacrylamide Monomer

A test sample (0.1 g) containing diethylaminoethylacrylamide is added tocarbon tetrachloride (2 mL). A 5% solution of bromine in carbontetrachloride is added drop by drop, with shaking, until the brominecolor persists. The bromine test is sensitive to the presence of C═Cbond, which can indicate the approximate content ofdiethylaminoethylacrylamide present in the test sample.

Example 4 Synthesis of an Ultra-Pure N-tris-hydroxymethylmethylacrylamide Monomer

An ultra-pure N-tris-hydroxymethyl methylacrylamide monomer wassynthesized according to the following scheme:

Briefly, acryloyl chloride was dissolved in CH₂Cl₂ and cooled to 0° C.N-(TrisHydroxy)methyl amine dissolved in CH₂Cl₂ was added drop wise atT_(i)=0-5° C. followed by NaOH (˜15% aq. solution). The reaction mixturewas stirred for 2 hours. The N-tris-hydroxymethyl methylacrylamide wasremoved after re-crystallization in water.

Example 5 HPLC Analysis of N-tris-hydroxymethyl methylacrylamide Monomer

First, a standard curve was prepared using N-[Tris (hydroxymethyl)methyl] acrylamide (trisacryl) with at least 98% purity (provided bySAFC; information from manufacturer's purity certificate).

Stock solution: approximately 200 mg (or 250 mg) of trisacryl wasweighed in a 200 mL (or 250 mL) reference flask and dissolved in purewater. The solution was then subjected to ultrasonication for 15 min andlet return to room temperature. The solution was adjusted to aconcentration of 1000 ppm (mg/L).

Intermediate solution: an intermediate solution was prepared by diluting5 ml of the 1000 ppm solution with water to a final volume of 50 mL.

The injected solutions are listed in Table 3. The exact concentrationsof the solutions were noted.

TABLE 3 Injected solutions Concentration Intermediate Total Solution(ppm) solution volume Water volume volume Standard 1 20 1 4 5 Standard 240 2 3 5 Standard 3 60 3 2 5 Standard 4 80 4 1 5 Standard 5 100 5 0 5

The samples to be analyzed were prepared as follows:

Stock solution: approximately 200 mg (or 250 mg) of trisacryl wasweighed in a 200 mL (or 250 mL) reference flask and dissolved in purewater. The solution was then subject to ultrasonication for 15 min andlet return to room temperature. The solution was adjusted to aconcentration of 1000 ppm (mg/L). Three solutions are prepared andtested.

Intermediate solution: an intermediate solution was prepared by diluting5 ml of the 1000 ppm solution with water to a final volume of 50 mL.

Sample solutions (each solution): sample solutions were prepared bydiluting the intermediate solutions to approximately 60 ppm (at least 5mL for sample injection). The intermediate solution (100 ppm) was alsotested. The exact concentrations were recorded.

The solutions (standards and samples) were then transferred to the HPLCvials on the support of the injector for analysis. The settings of theHPLC are listed in Table 4.

TABLE 4 HPLC settings Parameters Settings Injection Volume 20 μL OvenTemperature 30° C. Mobile phase Methanol/water 20/80 (0.2% TFA) Analysistime 6 min Wavelength 230 nm Column C18; 250 × 4.6 mm, 5 μm. Flow 1mL/min

The results of HPLC analysis showing the purity of trisacryl fromdifferent sources are shown in FIG. 1B. FIG. 1B depicts thesensitivities of purity for two different lots of trisacryl (R426 andR402 obtained from BioSepra) observed using a bromination reaction(right bars, as provided by manufacturer) or HPLC (left bars). The dataare shown with an assumption of 100% purity of a recrystallizedtrisacryl (TA-R) obtained and compared with trisacryl monomer from SAFCprior to recrystallization (TA) (see below). The TA-R was prepared byrecrystallizing TA (trisacryl from SAFC) in water and then dried. NMR ¹Hanalysis was performed on the recrystallized TA-R sample, and no organicimpurities were detected on the NMR ¹H spectrum (data not shown).Additionally, an elemental analysis was performed on the TA-R sample tocheck if there was inorganic salts present. The theoretical result for100% pure trisacryl is C, 47.99%, H, 7.48%, N, 8.00% and O, 36.53%. Itwas found for the TA sample obtained from SAFC: C, 46.76% H, 7.39% andN, 7.86% and TA-R 48.14%, H, 7.63% and N, 8.07%, so the assumption wasmade that the TA-R sample was close to 100% pure. Each of the R426, R402TA and TA-R samples were then tested by HPLC analysis, and the spectrawere recorded at 232 nm. For all the samples analyzed, there was goodlinearity between the pic area and the concentration (data not shown).With the assumption that TA-R was about 100% pure, the gradient of eachline was taken to define the HPLC purity of each trisacryl sample. Theresults of the HPLC analyses of the TA and TA-R samples are shown inFIG. 1B as discussed above.

Example 6 Bromine Test of N-tris-hydroxymethyl methylacrylamide Monomer

A test sample (0.1 g) containing N-tris-hydroxymethyl methylacrylamideis added to carbon tetrachloride (2 mL). A 5% solution of bromine incarbon tetrachloride is added drop by drop, with shaking, until thebromine color persists. The bromine test is sensitive to the presence ofC═C bond, which can indicate the approximate content ofN-tris-hydroxymethyl methylacrylamide present in the test sample.

Example 7 Microsphere Preparation Using an Ultra-PureN-tris-hydroxymethyl methylacrylamide Monomer

NaCl (58 g) and sodium acetate (27.2 g) were solubilized in water (300mL) under stirring in a one liter beaker. Glycerol (400 mL) was addedand the pH of the solution was adjusted to 6 with acetic acid. Thissolution was then heated at 60° C. under stirring and three monomerswere added to the solution. The monomer materials were not 100% pure andthe amount of each monomer used in the following steps was weighed withan adjustment of the amount of impurities present in the materials.Assuming that the monomers were 100% pure, the quantities of eachmonomer were as follows:

N,N-methylene-bis-acrylamide: 10 g.

Diethylaminoethylacrylamide: 35 g

N-tris-hydroxymethyl methylacrylamide: 90 g

Porcine gelatin (PB Leiner; Vilvoorde, Belgium): 20 g.

Diethylaminoethylacrylamide was supplied by Pall BioSepra, France. Thepurity of diethylaminoethylacrylamide was greater than 80% according toPall BioSepra's specification.

N-tris-hydroxymethyl methylacrylamide was supplied by Pall BioSepra andSigma Aldrich Fine Chemicals (SAFC)). According to suppliers' productspecification, the purity of N-tris-hydroxymethyl methylacrylamidesupplied by Pall BioSepra and SAFC is greater than 76% and 93%,respectively (see Table 5). Other comparative data ofN-tris-hydroxymethyl methylacrylamide supplied by PALL Biosepra and SAFCare provided in Table 5.

TABLE 5 Comparative data of N-tris-hydroxymethyl methylacrylamidesupplied by PALL Biosepra and SAFC Trisacryl Specification PALL BiosepraSAFC IR spectroscopy Conform to Conform to structure structure Moisture <5% No Purity >76% >93% Nitrogen content No C, H, N analysis; carbon44.6-51.4%, nitrogen 7.4-8.6% NMR Analysis No Conform to structure HPLCNo Yes Cl dosage No KCl <5%

When the solution was clear, a solution of gelatin (25 g) in water (250ml) was added to the monomer solution. This solution was filtered andplaced under stirring at 60° C. (Solution A). A solution of ammoniumpersulfate (3.5 g) in water (101.6 g) was also prepared (Solution B). Ina 10 liters beaker, oil (4 L) and Arlacel (3.1 g) were stirred at 60° C.(Solution C). Solution A and solution B were injected through pump Q2and pump RHO, respectively, in the Feed Ring equipment placed insolution C. After the injection was completed, the solution was stirredat 125 rpm for 30 minutes at 60° C. The reaction was stopped by adding acold solution of water, ice and surfactant. The microspheres obtainedwere washed several times with water until the oil was completelyremoved. The beaker was placed in an ultrasound bath for 10 minutes toreduce the number of sticking microspheres. The microspheres were thenheated at 37° C. and 300 ml of glutaraldehyde was added per 1 L ofmicrospheres for gelatin crosslinking. The microspheres were washedagain twice with water. The microspheres were then recovered bydecanting, washed carefully, screened and sterilized in an autoclave ina buffered medium.

Example 8 Microsphere Preparation Using an Ultra-PureN-tris-hydroxymethyl methylacrylamide Monomer and an UltrapureDiethylaminoethylacrylamide Monomer

NaCl (58 g) and sodium acetate (27.2 g) were solubilized in water (300mL) under stirring in a 1 liter beaker. Subsequently, glycerol (400 mL)was added and the pH of the solution was adjusted to 6 with acetic acid.

This solution was heated at 60° C. under stirring and two of the threemonomers were added to the solution. The monomer materials were not 100%pure and the amount of each monomer used in the following steps wasweighed with an adjustment of the amount of impurities present in thematerials. Assuming that the monomers were 100% pure, the quantities ofeach monomer were as follows:

N,N-methylene-bis-acrylamide: 10 g.

N-tris-hydroxymethyl methylacrylamide: 90 g

Diethylaminoethylacrylamide: 35 g

Porcine gelatin (PB Leiner; Vilvoorde, Belgium): 20 g.

N-tris-hydroxymethyl methylacrylamide was supplied by Pall BioSepra andSAFC. According to suppliers' product specification, the purity ofN-tris-hydroxymethyl methylacrylamide supplied by Pall BioSepra and SAFCis greater than 76% and 93%, respectively (see Table 5). Othercomparative data of N-tris-hydroxymethyl methylacrylamide supplied byPALL Biosepra and SAFC are provided in Table 5.

Diethylaminoethylacrylamide was supplied by PALL Biosepra and SAFC.According to suppliers' product specification, the purity ofdiethylaminoethylacrylamide supplied by Pall BioSepra and SAFC isgreater than 80% and 95%, respectively. Other comparative data ofdiethylaminoethylacrylamide supplied by PALL Biosepra and SAFC areprovided in Table 6:

TABLE 6 Comparative data of diethylaminoethylacrylamide supplied by PALLBiosepra and SAFC DEAE Acrylamide Specification PALL Biosepra SAFC IRspectroscopy Conform to structure Conform to structure Moisture  <5% NoPurity >80% >95% Nitrogen content 11-14% No NMR Analysis No Conform tostructure HPLC No Yes Gas Chromatography No Yes

The third monomer, diethylaminoethylacrylamide, was added after itspreparation. Diethylaminoethylacrylamide (35 g) was diluted with water(17 g). This solution was placed in an ice cold bath and the pH of thesolution was adjusted to 2 with HCl. This solution was added to thesolution containing the other two monomers.

When the solution was clear, gelatin (25 g) in water solution (250 mL)was added to the above monomer solution. This solution was filtered andplaced under stirring at 60° C. (solution A). Ammonium persulfate (3.5g) in water (101.6 g) was also prepared (Solution B). In a 10 litersbeaker, oil (4 L) and Arlacel (3.1 g) were stirred at 60° C. (SolutionC). Solution A and solution B were injected through pump Q2 and pumpRHO, respectively, in the Feed Ring equipment placed in solution C.After the injection was completed, the solution was stirred at 125 rpmfor 30 minutes at 60° C. The reaction was stopped by adding a coldsolution of water, ice and surfactant. The microspheres obtained werewashed several times with water until the oil was completely removed andthe beaker was placed in an ultrasound bath for 10 minutes to reduce thenumber of sticking microspheres. The microspheres were then heated at37° C. and 300 ml of glutaraldehyde was added per 1 L of microspheresfor gelatin crosslinking. The microspheres were washed again twice withwater. The microspheres were then recovered by decanting, washedcarefully, screened and sterilized in an autoclave in a buffered medium.

Example 9 Preparation of Colored Microsphere Using Ultra-Pure Monomers

Microspheres (100 mL) obtained according to Examples 7 or 8 above werewashed with a borate buffer (0.1 M, pH 8) and then suspended in anerythrosine isothiocyanate solution (50 mL, 5 mg/mL). The suspension wasthen stirred for at least 15 hours, after which it was washed with aneutral buffer to a colorless supernatant. The red-colored microsphereswere then calibrated and sterilized, and can be used in percutaneousembolization.

Example 10 Preparation of Opaque Preparation Using Ultra-Pure Monomers

The procedure of Examples 7 or 8 is followed, adding barium sulfatepower (200 g) to the initial monomer. The microspheres obtained areopaque to both visible light and x-rays.

Example 11 Preparation of Magnetic Microsphere Using Ultra-Pure Monomers

The procedure of Examples 7 or 8 is followed, adding 50 mg of magnetite(e.g., Fe₃O₄) to the initial monomer solution. Alternatively,microspheres obtained according to Examples 7 and 8 can each be packedinto a 16 mm diameter chromatographic column and washed with aphysiological buffer. The column is then loaded with a colloidalsuspension of ferrofluid (very small particles of magnetite) at a flowrate of 10 mL/hour. Particles of magnetite are adsorbed by themicrospheres and permanently trapped. Resulting microspheres can be usedfor regular embolization procedure and have magnetic properties, forexample, can be detected in Magnetic Resonance Imaging (MM) imagery.

Example 12 Separation of Non-Sticking Microspheres by Ultrasonication

Microspheres made from N-tris-hydroxymethyl methylacrylamide,diethylaminoethylacrylamide, N,N-methylene-bis-acrylamide and TEMED wereprocessed following different methods. Ultrasonication was done one ortwo times in a 35 KHz ultrasonic bath for approximately 10-15 minutes.

FIGS. 2A-2C illustrate the reduction of sticking microspheres byultrasonication. FIG. 2A shows microspheres before crosslinking,initially (left panel) and after 15 min of ultrasonication (rightpanel). The percentage of sticking microspheres decreases from about 7%to about 0.2% with ultrasonication. FIG. 2B demonstrates a second batchof microspheres (divided into two fractions) after crosslinking without(left panel) or with (right panel) ultrasonication prior tocrosslinking. Sieving was done after the ultrasonication step. Thepercentage of sticking microspheres decreases from about 3% to nearlyabout 0% with ultrasonication. FIG. 2C shows microspheres beforecrosslinking, initially (left panel) and after 2×15 min. ofultrasonication (right panel). The percentage of sticking microspheresdecreases from about 9.2% to about 1.6% with ultrasonication.

Taken together, in all cases, the microspheres showed less stickingspheres when ultrasonication is applied (FIGS. 2A-2C, right panels) ascompared to no ultrasonication (FIGS. 2A-2C, left panels). No brokenmicrospheres were observed by this method.

Table 7 and FIG. 3 depict results from a separate experiment and alsofurther illustrates reduced percentages of sticking microspheres (lotFM0904100) subject to ultrasonication before (FIG. 3A) and after gelatincrosslinking (FIG. 3B).

TABLE 7 Pour Pour Pour Pour Pour Day Day Day Day Day 1 2 3 4 5 MeanUltrasonic step Yes Yes Yes Yes Yes % of sticking spheres; 4.5 7.8 2.93.0 5.7 4.8 T450 before crosslinking % of sticking spheres; 0.2 0.4 0.71.1 0.3 0.3 T450 after crosslinking % of sticking spheres; 8.0 7.1 6.66.1 7.5 7.1 T630 before crosslinking % of sticking spheres; 0.2 0.4 0.71.1 0.3 0.6 T630 after crosslinking

Sieving was done after the ultrasonication step, and the percentage ofvolume on an 800 μm, 630 μm, 450 μm or 50 μm sieve is shown in Table 8and FIG. 3C.

TABLE 8 Pour Pour Pour Pour Pour Day Day Day Day Day 1 2 3 4 5 TotalT800 18 14 38 33 40 143 T630 16 14 35 37 30 132 T450 13 21 35 37 30 135T50  3 13 35 36 30   80 Total Volume 50 62 125  133  120  490 T800 36%23% 30% 25% 33% 29% T630 32% 23% 28% 28% 25% 27% T450 26% 34% 28% 27%25% 28% T50  6% 21% 14% 20% 17% 16%

Example 13 HR-MAS NMR Analysis of Monomers

The high resolution magic angle spinning (“HR-MAS”) technique wasemployed to analyze the starting materials used herein for microspheresynthesis.

Preparation of Samples

Trisacryl, porcine gelatin, DEAE-acrylamide and MBA samples (˜15 mgeach) were loaded in a HR-MAS rotor (see FIG. 7). Deuterated solvent wasthen added to obtain a total volume of 50 μL.

The rotors are referenced in Table 9.

TABLE 9 Rotor Sample Mass Solvent Total Volume (μL) 1.280 Trisacryl 15D₂O 50 1.281 Gelatin 12 D₂O 50 1.282 MBA 15 D₂O 50 1.274 DEAE-acrylamide15 D₂O 50

Analysis of Sample by NMR Spectroscopy

NMR spectra recorded on a Bruker Avance I spectrometer at 400 MHz (¹H)with a 4 mm HR-MAS probehead (¹H, ¹³C, ²H lock).

Tuning

After adjustment of the magic angle of the probehead, the followingadjustments were carried out for each sample:

Probehead tuning and matching (¹H)

90° pulse measurement

B₀ homogeneity adjustment (shims)

Acquisition

All experiments were performed at room temperature (298 K). For eachsample, a 1D ¹H NMR spectrum was recorded.

Processing

Acquisition and processing parameters are summarized in Table 10.

TABLE 10 Acquisition Parameters Spectrometer/Probehead Bruker Avance I,400 MHz (¹H)/HRMAS Temperature 25° C. Sample rotation 4000 Hz Locksubstance D₂O Pulse program zgcppr 90° ¹H pulse/(p1pl1) 6.35 μs/0 dBNumber of points (TD) 32k Acquisition time (AQ) 1.98 s Receiver gain(RG) 90.5 Relaxation delay (d₁) 1.5 sec Number of scans (NS) 4096Spectral width (SW/SWh) 20.69 ppm/1875.55 Hz Carrier frequency (O1p/O1)4.7 ppm/1878.55 Hz Processing parameters Number of points (SI) 32kApodization aucune Baseline correction Spline

Results

Typical signals for vinylic residues and aromatic moieties wereidentified in the spectra. The one dimensional ¹H NMR spectrum oftrisacryl, gelatin, MBA and DEAE acrylamide are shown in FIGS. 4A-4D.The attribution of ¹H nuclei for trisacryl, MBA and DEAE acrylamide areshown in FIGS. 4E-4G. The 1D ¹H NMR spectrum for gelatin consists ofpeaks from 7.20 to 7.30 ppm, which can be attributed, for example, tothe aromatic protons present in the amino acids.

Example 14 HR-MAS NMR Analysis of Microspheres

Microspheres prepared according to Example 8 using trisacryl and DEAEacrylamide from different sources (SAFC, PALL Biosepra) were analyzed byNMR spectroscopy.

Analysis of Sample by NMR Spectroscopy

Microsphere samples (˜3 mg each) made of trisacryl and DEAE acrylamidefrom different sources. The SAFC FMP 128 microsphere sample was labbench-prepared using trisacryl and DEAE obtained from SAFC (trisacryllot 1443257, 96% purity; DEAE lot 585803-199, 98% purity). The PALL FMP130 microsphere sample was lab-bench prepared using trisacryl and DEAEobtained from PALL Biosepra (trisacryl lot Y175; 90.9% purity; DEAE lotU089; 85% purity). The FM0903031-M1675 microsphere sample wasmanufactured using trisacryl obtained from SAFC (lot 03211DJ; 95.2%purity) and DEAE from PALL Biosepra (lot U089; 85.0% purity). TheFM0903021-M1654 microsphere sample was manufactured using trisacryl andDEAE obtained from PALL Biosepra (trisacryl lot W299, 88.2% purity; DEAElot U089; 85.0% purity). Purity levels were as provided bymanufacturers' specifications. Each of the four samples were loaded intoa rotor HR-MAS (see FIG. 7). Deuterated (D₂O) solvent was then added toobtain a total volume of 50 μL.

The rotors are referenced in Table 11.

TABLE 11 Rotor Sample Mass Solvent Total Volume (μL) 1.270 SAFC FMP 1283 D₂O 50 1.271 PALL FMP 130 3 D₂O 50

Tuning

After adjustment of the magic angle of the probehead, the followingadjustments were carried out for each sample:

Probehead tuning and matching (¹H)

90° pulse measurement

B₀ homogeneity adjustment (shims)

Acquisition

All experiments were performed at room temperature (25° C.). For eachsample, a one-dimensional ¹H NMR spectrum was recorded.

Processing

Acquisition and processing parameters are summarized in Table 12.

TABLE 12 Acquisition Parameters Spectrometer/Probehead Bruker Avance I,400 MHz (¹H)/HRMAS Temperature 25° C. Sample rotation 4000 Hz Locksubstance D₂O Pulse program zgcppr 90° ¹H pulse/(p1pl1) 6.35 μs/0 dBNumber of points (TD) 32k Acquisition time (AQ) 1.98 s Receiver gain(RG) 90.5 Relaxation delay (d₁) 1.5 sec Number of scans (NS) 4096Spectral width (SW/SWh) 20.69 ppm/1875.55 Hz Carrier frequency (O1p/O1)4.7 ppm/1878.55 Hz Processing parameters Number of points (SI) 32kApodization aucune Baseline correction Spline

Results

The results are shown in FIGS. 5A-5J. FIGS. 5A-5D illustrate the NMRspectra of samples SAFC FMP 128 and PALL FMP 130. FIGS. 5E-5J illustratethe NMR spectra of samples SAFC FMP 128, PALL FMP 130, FM0903031-M1675and FM0903021-M1654.

FIG. 5A is the superposition of the NMR spectra of two samples, whichshows a similarity between the corresponding main peaks (marked as A, Band C) identified in the spectra. The assignment of peaks A, B and C andother minor peaks were based on the analysis of HR-MAS NMR spectra ofstarting materials from Example 13.

Peak A: identified at 3.77 ppm, which may be attributed, for example, tothe tris-hydroxymethyl groups (C(CH₂OH)₃) in the copolymer.

Peak B: identified at 3.2 ppm, which may be attributed, for example, tothe CH2 groups linked to the basic nitrogen atom (CH₂N(CH₂CH₃)₂).Without wishing to be bound by any theory, the difference in thechemical shift between the pure monomers in D₂O and microspheressuggests that the amine is protonated (ammonium) in the crosslinkedstructure.

Peak C: identified at 1.3 ppm, which may be attributed, for example, tothe groups in the beta position of the carboxamide group of thepolymerized structure (CH₂—CHCONH). The relative integration ratios ofpeak B to peak A and peak C to peak A were carried out using asemi-quantitative approach by integration of the surface of the peaksand summarized in Table 13.

TABLE 13 Sample A B C PALL FMP 130 100 47.7 52.1 SAFC FMP 128 100 57.462.5

Without wishing to be bound by any theory, the higher integration ratiosof microspheres synthesized with purer trisacryl and DEAE acrylamide mayindicate that a better polymerization efficiency was achieved in thesynthesis compared to microspheres with less pure materials.

FIGS. 5B and 5C show the spectra in the regions of 9-5 ppm (5B) and 5-0ppm (5C). Residual peaks were identified between 5 and 9 ppm, centeredat 7.3 ppm, which may be attributed, for example, to the aromaticprotons present in gelatin. Peaks between 6.4 and 5.8 ppm may beattributed, for example, to traces of excess acrylamide during thepolymerization. FIG. 50 and Table 13 show the slight difference in thechemical shift (δ) for peaks B and C.

FIG. 5E shows a comparison of the ¹H NMR spectra of four differentsamples. A triplet at 1.15 ppm and a quadruplet at 3.62 ppm wereidentified from the FM0903031-M1675 and FM0903021-M1654 spectra. Asinglet at 2.2 ppm was identified from the FM0903031-M1675 spectrum,which is also present in the SAFC 128 and PALL FMP 130 spectra but withmuch less intensity (See FIG. 5F).

These resonance lines are relatively thin compared to the main peak,which may be due to the presence of small organic molecules. The tripletat 1.15 ppm and quadruplet at 3.62 ppm may be attributed, for example,to the presence of ethanol or ether diethyl. The chemical shifts ofpeaks B and C are identical for FM0903031-M1675 and FM0903021-M1654,which are different from PALL FMP 130 and SAFC FMP 128 (FIG. 5G). Table14 summarizes the differences in the observed chemical shifts.

TABLE 14 Sample δ_(B) (ppm) δ_(C) (ppm) Δ(δ_(B)) (Hz)* Δ(δc) (Hz)* PALLFMP 130 3.212 1.284 0 0 SAFC FMP 128 3.192 1.276 8.00 3.20FM0903031-M1675 3.246 1.298 −13.60 −5.60 FM0903021-M1654 3.246 1.298−13.60 −5.60 *Difference in Hz measured relative to the the resonancesof peaks B and C of the sample PALL FMP 130*Difference in Hz measured relative to the resonances of peaks B and Cof the sample PALL FMP 130

A peak located at 8.4 ppm was identified in the FM0903031-M1675 andFM0903021-M1654 spectra (FIG. 5H).

Semi-quantitative approach was employed to analyze the spectra. Thesuperposition of the four spectra (FIG. 5I) illustrates the variationsin the intensity and width at half height of peaks B and C. Adeconvolution of mass A, B, and C was performed for each spectrum forquantification (FIG. 5J) using computer software. The amplitude valuesof peaks B and C calculated and normalized to the peak area A aresummarized in Table 15.

TABLE 15 Sample A B C PALL FMP 130 100 47.7 52.1 SAFC FMP 128 100 57.462.50 FM0903031-M1675 100 49.5 62.30 FM0903021-M1654 100 49.7 60.90

Without wishing to be bound by any theory, the differences observed forpeaks B and C among different samples can be explained, in part, asfollows: Differences in the chemical shifts and widths at half heightmay be attributed to residual degrees of anisotropy between differentsamples. Even if the swelling in water combined with the rotation magicangle allows to average the anisotropy of displacement chemical anddipolar couplings, these effects are not completely canceled. Theseresidual effects observable for the NMR spectra may be attributed to thedegree of crosslinking in different samples. Different surfaces may beexplained by the differences in the microstructure of the polymers. Thedifferences in the presence or absence of small organic molecules may beexplained by a difference in the manufacturing process or by adifference in the materials used. The differences in the chemical shiftand width at half height observed for peaks B and C can be used todifferentiate the sources of the samples.

Example 15 Administration of Microspheres in Patients with Liver Cancers

This study is carried out according to a modified procedure described inXu et al. (2009) World J Gastroenterol 15(29): 3664-3669. Briefly,embolization (optionally TACE) is performed in patients aftercross-sectional images are reviewed. A French vascular sheath is placedinto the femoral artery, and a Mickaelson catheter is advanced into theceliac and superior mesenteric arteries. Contrast is injected into thearteries during rapid-sequence radiographic imaging. Arterial branchessupplying the tumors are then located. The venous phase is carefullyexamined for patency of the portal veins. A Tracker catheter is advancedthrough the Mickaelson catheter to the arterial branches supplying thetumors. The mixture of doxorubicin (˜50 mg), mitomycin (˜10 mg), andlipiodol (˜4-15 mL) is injected into the arterial branches untilhemostasis was achieved. If the tumors have no shrinkage 2 weeks afterthe procedure, a second embolization can be performed.

The interventional radiologist then performs an arteriogram to identifythe branches of the hepatic artery supplying the tumor(s) and threadssmaller catheters into these branches. This is done to maximize theamount of the chemotherapeutic dose that is directed to the tumor.

When a blood vessel supplying tumor has been selected, microspheresalone or, in the case of TACE, alternating aliquots of the chemotherapydose and of microspheres prepared according to Examples 7 or 8 (or othermicrosphere provided herein), or microspheres in combination with thechemotherapy agent, are injected through the catheter. The totalmicrosphere and/or chemotherapeutic dose may be given in one vessel'sdistribution, or it may be divided among several vessels supplying thetumor(s).

Example 16 Administration of Microspheres in Patients with ProstateCancer

Patients diagnosed with prostate cancer are divided up into two groups,a test group and a control group. Patients from the test group aretreated with embolization, using microspheres prepared according toExample 7 or 8 (or other microspheres provided herein).

Biopsies are performed in patients with visible prostate tumors.Patients whose prostate tumors are diagnosed to be cancers are treatedwith embolization. Before treatment, sizes of the prostate cancer tumorsand levels of the blood PSA in patients having the prostate cancertumors are recorded.

Prior to the embolization procedure, a prophylactic single dose of 200mg ciprofloxacin is given. Intervention is performed under localanesthesia through the right transfemoral approach. Initial pelvicangiography is performed to evaluate the iliac vessels and prostatearteries during the arterial and late phases. Selective digitalsubtraction angiography of the right and left internal iliac arteries isperformed using a 5-Fr Cobra 2 catheter to better assess the bloodsupply to the prostate. Superselective catheterization of the right andleft inferior vesicle arteries is then performed using a microcatheter(Embocath; Biosphere Medical, Rockland, Me., USA) and angiography isperformed by manually injecting 3-5 ml of contrast medium to ensure thatthe tip of the microcatheter is inside or at the ostium of the prostaticarteries. Microspheres prepared according to Examples 7 or 8 arecalibrated to 300-500 μm in diameter are used for embolization. Each2-ml vial of microspheres is diluted in a mixture of 20 ml of 50%iodinated contrast medium plus 50% normal saline solution. The mixtureis slowly injected under fluoroscopic guidance. Embolization of theprostate arteries arising from the inferior vesical arteries isperformed to stasis without reflux of the mixture to undesired arteries.Follow-up angiography is performed manually with the microcatheter atthe inferior vesical artery and using the pump with the 5-Fr catheter atthe anterior branch of the internal iliac artery checking any furtherblood supply to the prostate. Embolization is then performed on theother side using the same technique.

Weekly measurements of the tumor size are recorded. Tumor volume iscalculated (tumor volume=0-5 (L+W)×L×W×.0.5236, where L=tumor length andW=width). The tumor volume at the time of embolization is served as thepoint of reference for future comparison of that tumor's size variation.The weekly variations of each tumor volume are recorded as percentdifferentiation from the original measurement at embolization. The bloodPSA levels are followed and compared with the levels at the time ofembolization.

Example 17 Administration of Microspheres in Patients with ArteriovenousMalformation

Patients with brain arteriovenous malformations (BAVM) receiveembolization according to a modified procedure described Gao et al.(2009) Chinese Medical Journal 122(16):1851-1856. Briefly, embolizationis carried out under general anesthesia without systemic heparinization.During the procedure, systolic blood pressure is controlled between 100and 110 mmHg. Catheterization is performed using a transfemoral approachwith standard coaxial techniques. The guiding catheter is flushed via apressure bag with saline containing 2500 U heparin/L. Navigation of aDMSO-compatible microcatheter (UltraFlow or Marathon; ev3; USA) to thetarget is performed with a combination of the flow-guided andwire-guided techniques using a 0.010-inch or a 0.008-inch wire(Silverspeed or Mirage; ev3; USA). Once the microcatheter tip is in thedesired position, the injection of microspheres prepared according toExample 7 or 8 (or other microspheres provided herein) is carried out asfollows: 1) the microcatheter is flushed with 5 ml normal saline; 2)0.25 ml DMSO is injected into the microcatheter to fill the dead space;3) microspheres aspirated into a 1 ml syringe, and 0.25 ml of thisamount is injected over for 40 seconds to fill the microcatheter and toreplace the DMSO in the dead space; 4) slow injection of the microspheresolution is then continued under fluoroscopy.

After embolization, patient blood pressure is strictly monitored for 48hours in the intensive care unit. In rare event the microcathetersticking in the arterial feeder, heparin is intravenously administered(750-1000 U/hour) for 72 hours, followed by oral aspirin for 3 months ata dose of 250 mg/d. The BAVM nidus size is then monitored by yearly MM.If after three years the AVM is still not obliterated, radiosurgery isconsidered.

Example 18 Administration of Microspheres in a Porcine Model of a BenignProstatic Hyperplasia

The study is carried out according to a modified procedure described inSun et al. (2008) Radiology 246: 783-789. Briefly, pigs are randomlyassigned to the embolization group or the control group. After fastingfor 24 hours, all male pig are each premedicated with 0.1 mg of diazepamper kilogram of body weight, 10 mg/kg ketamine, and 0.01 mg/kg atropineintramuscularly. Anesthesia is initiated with 2 mg/kg propofol (i.v.)and maintained with 2.0%-2.5% halothane. Each pig is connected to ananesthesia system and a mechanical ventilator after endotracheallyintubated. The groin area and lower abdomen were shaved and draped in asterile manner.

Femoral arterial access is gained percutaneously with a 5-F introducersheath. Pelvic angiography is performed, a 5-F Cobra catheter isinserted into the aorta and the catheter tip is shaped into a Waltmanloop from the contralateral external iliac artery. With road mapguidance, selective catheterization to the internal iliac artery on bothsides is achieved. Angiography is performed in the two arteries. Thecontrol group animals then recovered from anesthesia.

Selective embolization of the prostate is performed in the embolizationgroup. After the systemic distribution of heparin (150 IU ofheparin/kilogram of body weight), a 3-F infusion catheter is insertedcoaxially through the Cobra catheter and selectively placed in theprostatic branch of the inferior vesical artery. Superselectiveangiography is performed by manually injecting 1 mL of contrast mediumto ensure that the tip of the microinfusion catheter is at the desiredsite. Microspheres prepared according to Example 7 or 8 (or othermicrospheres provided herein) are calibrated to 500-700 μm in diameter.Each vial of microspheres, containing 2.0 mL of microsphere particles,is diluted in a mixture of 20 mL of 50% iodinated contrast medium plus50% normal saline solution. The mixture is slowly injected withfluoroscopic guidance. Embolization is immediately terminated oncehemostasis is achieved without reflux of the mixture to undesiredarteries. Follow-up angiography is performed. Subsequently, embolizationis performed on the other side by using the same technique. The animalsare allowed to recover from anesthesia under care. After embolization,all animals are checked twice a day for 72 hours and then once daily for1 week for possible complications associated with embolization.

Example 19 Administration of Microspheres in Patients with UterineFibroids

Uterine artery embolization (UAE) is carried out according to a modifiedprocedure described in Pelage et al. (2000) Radiology 215:428-431.Briefly, UAE is performed by an interventional radiologist and involvescomplete occlusion of either one or both uterine arteries withparticulate emboli to cause ischaemic necrosis of the uterine fibroids.The closure of the arteries is considered permanent, thereby blockingblood supply to the fibroid but without any permanent adverse effect onthe otherwise normal uterus. UAE is performed under local anaesthetic,sometimes with conscious sedation, epidural or spinal anaesthetic.Prophylactic antibiotics may also be administered. A vascular sheath of4 or 5 French diameter is inserted directly into the woman's femoralartery and the contralateral uterine artery is then selectivelycatheterized. The catheter may then be maneuvered to the ipsilateraluterine artery and the process repeated.

In this study, microspheres prepared according to Example 7 or 8 (orother microspheres provided herein) are calibrated to 700-900 μm indiameter and administered. Occlusion of the uterine vessels is confirmedby angiography and the catheter removed. The woman is exposed toapproximately 20 rad (20 cGy) of ionizing radiation to the ovaries.Successful UAE totally occludes both uterine vessels. The normalmyometrium (muscle of the womb) rapidly establishes a new blood supplythrough collateral vessels from the ovarian and the vaginalcirculations.

The embodiments of the present invention described above are intended tobe merely exemplary, and those skilled in the art will recognize, or beable to ascertain using no more than routine experimentation, numerousequivalents to the specific procedures described herein. All suchequivalents are considered to be within the scope of the presentinvention and are covered by the following claims. Furthermore, as usedin this specification and claims, the singular forms “a,” “an” and “the”include plural forms unless the content clearly dictates otherwise.Thus, for example, reference to “a microsphere” includes a mixture oftwo or more such microspheres. Additionally, ordinarily skilled artisanswill recognize that operational sequence must be set forth in somespecific order for the purpose of explanation and claiming, but thepresent invention contemplates various changes beyond such specificorder.

What is claimed is:
 1. A method of making microspheres, comprising:preparing an aqueous solution comprising one or more monomers; andadding the aqueous solution into a liquid organic phase using a feedsystem, before or while stirring, thereby producing microspherescomprising a polymer or copolymer comprising one or more monomer units,wherein the feed system comprises feed equipment that is disposed in theliquid organic phase, and wherein the aqueous solution is passed throughthe feed equipment and injected into the liquid organic phase.
 2. Themethod of claim 1, wherein the feed system comprises feed ring equipmentthat is placed in the liquid organic phase, and wherein the step ofadding the aqueous solution into the liquid organic phase comprisespassing the aqueous solution through the feed ring equipment into theliquid organic phase.
 3. The method of claim 1, wherein the feed systemcomprises a longitudinal axis and a plurality of spaced fingersextending outward from a cross-member connected to the longitudinalaxis.
 4. The method of claim 2, wherein the feed system comprises afirst pump and a second pump, wherein the aqueous solution is injectedthrough the first pump into the feed ring equipment and wherein anaqueous ammonium persulfate solution is injected through the second pumpinto the feed ring equipment.
 5. The method of claim 4, wherein theaqueous solution is injected through the first pump into the feed ringequipment and the aqueous ammonium persulfate solution is injectedthrough the second pump into the feed ring equipment simultaneously. 6.The method of claim 1, wherein the aqueous solution further comprisesgelatin, and wherein the method further comprises a step of crosslinkingthe gelatin.
 7. The method of claim 1, further comprising: subjectingthe microspheres to ultrasonication.
 8. The method of claim 1, whereinthe method does not comprise sieving the microspheres.
 9. The method ofclaim 1, wherein the liquid organic phase has 15% or less miscibility inwater at 25° C.
 10. The method of claim 1, wherein the liquid organicphase comprises an oil.
 11. The method of claim 1, wherein the one ormore monomers comprise an acrylamide monomer.
 12. The method of claim11, wherein the acrylamide monomer comprises at least one ofN-tris-hydroxymethyl methylacrylamide, diethylaminoethylacrylamide, orN,N-methylene-bis-acrylamide.
 13. The method of claim 1, wherein themicrospheres have a diameter from about 1 μm to about 2000 μm.
 14. Themethod of claim 1, wherein less than 1% of the microspheres areaggregated microspheres.
 15. Microspheres prepared by the method ofclaim
 1. 16. A method of making microspheres, comprising: preparing anaqueous solution comprising one or more monomers; and adding the aqueoussolution into a liquid organic phase using a feed system, before orwhile stirring, thereby producing microspheres comprising a polymer orcopolymer comprising one or more monomer units, wherein the feed systemcomprises a longitudinal axis and a plurality of spaced fingersextending outward from a cross-member connected to the longitudinalaxis, wherein the aqueous solution is passed through the feed system andinjected into the liquid organic phase.
 17. The method of claim 16,wherein the liquid organic phase comprises an oil.
 18. The method ofclaim 16, wherein the one or more monomers comprise an acrylamidemonomer.
 19. A method of making microspheres, comprising: preparing anaqueous solution comprising one or more monomers; and adding the aqueoussolution into a liquid organic phase using a feed system, before orwhile stirring, thereby producing microspheres comprising a polymer orcopolymer comprising one or more monomer units, wherein the feed systemcomprises feed ring equipment, wherein the aqueous solution is passedthrough the feed ring equipment and into the liquid organic phase. 20.The method of claim 19, wherein the one or more monomers comprise anacrylamide monomer.